Pyrazolopyridazines and methods for treating retinal-degenerative diseases and hearing loss associated with usher syndrome

ABSTRACT

Compounds, compositions and methods for the treatment of retinal degenerative diseases, such as retinitis pigmentosa, Leber&#39;s congenital Amaurosis, Syndromic retinal degenerations, age-related macular degeneration and Usher Syndrome, and hearing loss associated with Usher Syndrome are described herein.

This application is a continuation of U.S. application Ser. No.14/731,535, filed Jun. 5, 2015, which is a division of U.S. applicationSer. No. 13/791,744, filed Mar. 8, 2013, now U.S. Pat. No. 9,227,976,which claims the benefit of U.S. Provisional Application No. 61/718,611,filed Oct. 25, 2012, the disclosure of each of which is herebyincorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Usher Syndrome, a rare genetic disorder and a leading cause of deafnessand blindness, is associated with a mutation in any one of ten genes.Other names for the syndrome include Hallgren Syndrome, Usher-HallgrenSyndrome, RP-Dysacusis Syndrome, and Dystrophia Retinae DysacusisSyndrome.

Usher Syndrome is characterized by deafness and gradual vision loss. Thehearing loss is associated with inner ear defects, whereas the visionloss is associated with retinitis pigmentosa (RP), a degeneration of theretinal cells. Usually, the rod cells of the retina are affected first,leading to early night blindness and the gradual loss of peripheralvision. Some cases involve early degeneration of the cone cells of themacula, leading to a loss of central acuity. In some cases, thesufferer's foveal vision is spared, leading to “doughnut vision,” inwhich central and peripheral vision remain intact, but interrupted by aring of blindness.

Usher Syndrome has three clinical subtypes, denoted: I, II and III.Usher I subjects are born profoundly deaf, begin to lose vision withinten years and exhibit balance difficulties. They are slow to learn towalk as children, due to vestibular abnormalities. Usher II subjectssuffer lesser hearing loss, do not suffer physical imbalance and beginto lose vision in adolescence. Much of their hearing can be preservedinto middle age. Usher III subjects suffer gradual loss of hearing andvision and can suffer physical imbalance.

Usher Syndrome is a variable condition; the degree of severity is nottightly linked to subtype. For example, an Usher III subject might beasymptomatic in childhood, but develop profound hearing and vision lossby early to mid adulthood. Substantial visual impairment prior to age 50is common in Usher III subjects. An Usher I subject, on the other hand,might be deaf from birth, but sustain good central vision into old age.

SUMMARY OF THE INVENTION

The invention provides compounds of Formula I:

and pharmaceutically acceptable salts thereof,

wherein R₁ is:

R₂ is:

and

a is 0, 1, or 2.

The invention also provides compounds of Formula II:

and pharmaceutically acceptable salts thereof,

wherein R₃ is:

b is 0 or 1; and

c is 1 or 2.

The invention additionally provides compounds of Formula III:

and pharmaceutically acceptable salts thereof,

wherein R₄ is

The invention further provides compounds of Formula XIII:

and pharmaceutically acceptable salts thereof,

wherein R₅ is:

R₆ is:

and

a is 0, 1, or 2.

The invention also provides compounds of Formula XIV:

and pharmaceutically acceptable salts thereof,

wherein R₇ is:

b is 0 or 1; and

c is 1 or 2.

The invention also provides compounds of Formula XV:

and pharmaceutically acceptable salts thereof,

wherein R₈ is:

The invention further provides the following Pyrazolopyridazinecompounds:

and pharmaceutically acceptable salts thereof.

A compound of Formula I, II, III, XIII, or XIV, Compound 20-30 or 31, ora pharmaceutically acceptable salt thereof, (a “Pyrazolopyridazinecompound” or a “compound of the invention”) is useful for treating aretinal degenerative disease or hearing loss associated with UsherSyndrome.

The invention further provides compositions comprising an effectiveamount of a Pyrazolopyridazine compound and a pharmaceuticallyacceptable carrier or vehicle. The compositions are useful for treatinga retinal degenerative disease or hearing loss associated with UsherSyndrome.

The invention further provides methods for treating a retinaldegenerative disease, comprising administering to a subject in needthereof an effective amount of a Pyrazolopyridazine compound.

The invention still further provides methods for treating hearing lossassociated with Usher Syndrome, comprising administering to a subject inneed thereof an effective amount of a Pyrazolopyridazine compound.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 illustrates density of N48K Clarin-1 expression in cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compounds of the invention, compositionscomprising a compound of the invention, and methods for treating aretinal degenerative disease or hearing loss associated with UsherSyndrome, comprising administering a Pyrazolopyridazine compound or apharmaceutically acceptable salt thereof.

Compounds of the Invention

The word “about” when immediately preceding a numerical value means arange of plus or minus 10% of that value, e.g., “about 100 mg” means 90mg to 110 mg, “about 300 mg” means 270 mg to 330 mg, etc.

Abbreviations:

-   APCI Atmospheric Pressure Chemical Ionization-   DAPI 4′,6-diamidino-2-phenylindole-   DIPEA diisopropylethylamine-   DMEM Dulbecco's Modified Eagle Medium-   DMF dimethylformamide-   DMSO Dimethyl sulfoxide-   EDAC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride-   ESI Electrospray ionization-   ESI-TOF Electrospray ionization-Time-of-flight-   HATU 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   HOPO 2-hydroxypyridine-N-oxide-   HPLC High-performance liquid chromatography-   LCMS Liquid chromatography-mass spectrometry-   LDA lithium diisopropyl amide-   m/z Mass-to-charge ratio-   MALDI-TOF Matrix Assisted Laser Desorption Ionization-Time-of-flight-   MS Mass spectrometry-   PBS phosphate-buffered saline-   Rt Retention time-   SDS sodium dodecylsulfate-   THF tetrahydrofuran    Compounds of Formula I

In one embodiment, the Pyrazolopyridazine compound is a compound ofFormula I:

or a pharmaceutically acceptable salt thereof,wherein R₁ is:

R₂ is:

and a is 0, 1, or 2.

In particular embodiments, R₁ is —I. In other embodiments, R₁ is —H. Inyet other embodiments, R₁ is —CH₃. In certain embodiments, R₁ is —CF₃.

In yet other embodiments, R₁ is

In certain embodiments, R₁ is

In still further embodiments, R₁ is

In particular embodiments, R₁ is

In other embodiments, R₁ is

In yet other embodiments, R₁ is

In certain embodiments, R₁ is

In particular embodiments, R₁ is

In certain embodiments, R₁ is

In still further embodiments, R₁ is

In other embodiments, R₁ is

In yet other embodiments, R₁ is

In certain embodiments, R₁ is

In still further embodiments, R₁ is

In other embodiments, R₁ is

In particular embodiments, R₁ is

In further embodiments, R₁ is

In still further embodiments, R₁ is

In certain embodiments, R₂ is —H. In yet other embodiments, R₂ is

In particular embodiments, R₂ is

In yet other embodiments, R₂ is

In further embodiments, R₂ is

a=1, and Hal is —F.

In certain embodiments, R₂ is

In still further embodiments, R₂ is

In particular embodiments, R₂ is

In other embodiments, R₂ is

In yet other embodiments, R₂ is

In certain embodiments, R₂ is

In further embodiments, when a is 2, each Hal is the same or different.

Illustrative compounds of Formula I are:

and pharmaceutically acceptable salts thereof.Compounds of Formula II

The invention also provides compounds of Formula II:

and pharmaceutically acceptable salts thereof,

-   wherein R₃ is:

and

-   b is 0 or 1; and-   c is 1 or 2.

In particular embodiments, b is 0. In other embodiments b is 1 and the—F is in the meta position relative to the pyrazolopyridazino ringsystem. In yet other embodiments b is 1 and the —F is in the paraposition relative to the pyrazolopyridazino ring system.

In particular embodiments R₃ is —CF₃. In certain embodiments R₃ is

In other embodiments R₃ is

In yet other embodiments R₃ is

In further embodiments R₃ is

In still further embodiments R₃ is

In particular embodiments R₃ is

In other embodiments R₃ is

In yet other embodiments R₃ is

In certain embodiments R₃ is

In further embodiments R₃ is

In further embodiments R₃ is

In certain embodiments R₃ is

In other embodiments R₃ is

In yet other embodiments R₃ is

In further embodiments R₃ is

In still further embodiments R₃ is

In particular embodiments R₃ is

In certain embodiments R₃ is

In further embodiments R₃ is

and c=1. In still further embodiments R₃ is

In particular embodiments R₃ is

In other embodiments R₃ is

and c=2. In yet other embodiments R₃ is

In certain embodiments R₃ is

In other embodiments R₃ is

In yet other embodiments R₃ is

Illustrative compounds of Formula II are:

and pharmaceutically acceptable salts thereof.Compounds of Formula III

The invention additionally provides compounds of Formula III:

and pharmaceutically acceptable salts thereof,

wherein R₄ is

In certain embodiments R₄ is

In particular embodiments R₄ is

In other embodiments R₄ is

In yet other embodiments R₄ is

Illustrative compounds of Formula III are:

and pharmaceutically acceptable salts thereof.Compounds of Formula XIII

The invention provides compounds of Formula XIII:

and pharmaceutically acceptable salts thereof,

wherein R₅ is:

R₆ is:

and

a is 0, 1, or 2.

In particular embodiments, R₅ is —I. In other embodiments, R₅ is —H. Inyet other embodiments, R₅ is —CH₃. In certain embodiments, R₅ is —CF₃.

In yet other embodiments, R₅ is

In certain embodiments, R₅ is

In still further embodiments, R₅ is

In particular embodiments, R₅ is

In other embodiments, R₅ is

In yet other embodiments, R₅ is

In certain embodiments, R₅ is

In particular embodiments, R₅ is

In certain embodiments, R₅ is

In still further embodiments, R₅ is

In other embodiments, R₅ is

In yet other embodiments, R₅ is

In certain embodiments, R₅ is

In still further embodiments, R₅ is

In other embodiments, R₅ is

In particular embodiments, R₅ is

In further embodiments, R₅ is

In still further embodiments, R₅ is

In certain embodiments, R₅ is

In further embodiments, R₅ is

In further embodiments, R₅ is

In other embodiments, R₅ is

In yet other embodiments, R₅ is

In particular embodiments, R₅ is

In further embodiments, R₅ is

In still further embodiments, R₅ is

In certain embodiments, R₅ is

In other embodiments, R₅ is

In yet other embodiments, R₅ is

In particular embodiments, R₅ is

In further embodiments, R₅ is

In still further embodiments, R₅ is

In certain embodiments, R₅ is

In other embodiments, R₅ is

In yet other embodiments, R₅ is

In particular embodiments, R₅ is

In further embodiments, R₅ is

In still further embodiments, R₅ is

In certain embodiments, R₅ is

In certain embodiments, R₆ is

In further embodiments, R₆ is

and a=0. In other embodiments, R₆ is

In yet other embodiments, R₆ is

In particular embodiments, R₆ is

In certain embodiments, R₆ is

In further embodiments, R₆ is

In other embodiments, R₆ is

In certain embodiments, R₆ is

In further embodiments, R₆ is

In particular embodiments, R₆ is

In further embodiments, R₆ is

In still further embodiments, R₆ is

In other embodiments, R₆ is

In certain embodiments, R₆ is

In yet other embodiments, R₆ is

In particular embodiments, R₆ is

In further embodiments, R₆ is

In still further embodiments, R₆ is

In certain embodiments, R₆ is

In other embodiments, R₆ is

In further embodiments, when a is 2, each Hal is the same or different.

Illustrative compounds of Formula XIII are:

and pharmaceutically acceptable salts thereof.Compounds of Formula XIV

The invention also provides compounds of Formula XIV:

and pharmaceutically acceptable salts thereof,

wherein R₇ is:

b is 0 or 1; and

c is 1 or 2.

In particular embodiments, b is 0. In other embodiments b is 1 and the—F is in the meta position relative to the pyrazolopyridazino ringsystem. In yet other embodiments b is 1 and the —F is in the paraposition relative to the pyrazolopyridazino ring system.

In particular embodiments R₇ is —CF₃. In certain embodiments R₇ is

In other embodiments R₇ is

In yet other embodiments R₇ is

In further embodiments R₇ is

In still further embodiments R₇ is

In particular embodiments R₇ is

In other embodiments R₇ is

In yet other embodiments R₇ is

In certain embodiments R₇ is

In further embodiments R₇ is

In further embodiments R₇ is

In certain embodiments R₇ is

In other embodiments R₇ is

In yet other embodiments R₇ is

In further embodiments R₇ is

In still further embodiments R₇ is

In particular embodiments R₇ is

In certain embodiments R₇ is

In further embodiments R₇ is

and c=1. In still further embodiments R₇ is

In particular embodiments R₇ is

In other embodiments R₇ is

and c=2. In yet other embodiments R₇ is

In certain embodiments R₇ is

In other embodiments R₇ is

In yet other embodiments R₇ is

In yet other embodiments R₇ is

An illustrative compound of Formula XIV is:

and pharmaceutically acceptable salts thereof.Compounds of Formula XV

The invention also provides compounds of Formula XV:

and pharmaceutically acceptable salts thereof,

wherein R₈ is:

In particular embodiments R₈ is

In certain embodiments R₈ is

In other embodiments R₈ is

In yet other embodiments R₈ is

In further embodiments R₈ is

In still further embodiments R₈ is

In particular embodiments R₈ is

In particular embodiments R₈ is

In certain embodiments R₈ is

In other embodiments R₈ is

In yet other embodiments R₈ is

In further embodiments R₈ is

In still further embodiments R₈ is

Illustrative compounds of Formula XV are:

and pharmaceutically acceptable salts thereof.Additional Pvrazolopyridazine CompoundsThe invention further provides the following Pyrazolopyridazinecompounds:

and pharmaceutically acceptable salts thereof

Some of the compounds disclosed herein, for example, Compounds Ip, Iq,It, IIj, IIt, IIu, IIx, IIy, XIIIe, XIIIf, XIIIg, XIIIh. XIIIi; XIIIv,and XIIIw; are depicted having a bold or hatched wedge, indicatingabsolute stereochemistry.

Without being bound by any particular mechanism, it is believed that thebisphenyl pyrazolopyridazine moiety of Pyrazolopyridazine compounds isinvolved in the restoration of the activity and trafficking of Clarin I,which is the protein encoded by the gene mutated in Usher III Syndrome(Adato et al., Eur J Hum Genet. 2002 June; 10(6):339-50)

The compounds of the invention can be in the form of a salt. In someembodiments, the salt is a pharmaceutically acceptable salt.Pharmaceutically acceptable salts include, for example, acid-additionsalts and base-addition salts. The acid that forms an acid-addition saltcan be an organic acid or an inorganic acid. A base that forms abase-addition salt can be an organic base or an inorganic base. In someembodiments, a pharmaceutically acceptable salt is a metal salt. In someembodiments, a pharmaceutically acceptable salt is an ammonium salt.

Acid-addition salts can arise from the addition of an acid to thefree-base form of a compound of the invention. In some embodiments, theacid is organic. In some embodiments, the acid is inorganic.Non-limiting examples of suitable acids include hydrochloric acid,hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuricacid, sulfurous acid, a phosphoric acid, nicotinic acid, isonicotinicacid, lactic acid, salicylic acid, 4-aminosalicylic acid, tartaric acid,ascorbic acid, gentisinic acid, gluconic acid, glucaronic acid, saccaricacid, formic acid, benzoic acid, glutamic acid, pantothenic acid, aceticacid, propionic acid, butyric acid, fumaric acid, succinic acid, citricacid, oxalic acid, maleic acid, hydroxymaleic acid, methylmaleic acid,glycolic acid, malic acid, cinnamic acid, mandelic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, phenylaceticacid, N-cyclohexylsulfamic acid, methanesulfonic acid, ethanesulfonicacid, benzenesulfonic acid, p-toluenesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 2-phosphoglyceric acid,3-phosphoglyceric acid, glucose-6-phosphoric acid, and an amino acid.

Non-limiting examples of suitable acid-addition salts include ahydrochloride salt, a hydrobromide salt, a hydroiodide salt, a nitratesalt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt,a hydrogen phosphate salt, a dihydrogen phosphate salt, a carbonatesalt, a bicarbonate salt, a nicotinate salt, an isonicotinate salt, alactate salt, a salicylate salt, a 4-aminosalicylate salt, a tartratesalt, an ascorbate salt, a gentisinate salt, a gluconate salt, aglucaronate salt, a saccarate salt, a formate salt, a benzoate salt, aglutamate salt, a pantothenate salt, an acetate salt, a propionate salt,a butyrate salt, a fumarate salt, a succinate salt, a citrate salt, anoxalate salt, a maleate salt, a hydroxymaleate salt, a methylmaleatesalt, a glycolate salt, a malate salt, a cinnamate salt, a mandelatesalt, a 2-phenoxybenzoate salt, a 2-acetoxybenzoate salt, an embonatesalt, a phenylacetate salt, an N-cyclohexylsulfamate salt, amethanesulfonate salt, an ethanesulfonate salt, a benzenesulfonate salt,a p-toluenesulfonate salt, a 2-hydroxyethanesulfonate salt, anethane-1,2-disulfonate salt, a 4-methylbenzenesulfonate salt, anaphthalene-2-sulfonate salt, a naphthalene-1,5-disulfonate salt, a2-phosphoglycerate salt, a 3-phosphoglycerate salt, aglucose-6-phosphate salt, and an amino acid salt.

Metal salts can arise from the addition of an inorganic base to acompound of the invention having a carboxyl group. The inorganic baseconsists of a metal cation paired with a basic couterion, such as, forexample, hydroxide, carbonate, bicarbonate, or phosphate. The metal canbe an alkali metal, alkaline earth metal, transition metal, or maingroup metal. Non-limiting examples of suitable metals include lithium,sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium,strontium, cobalt, titanium, aluminum, copper, cadmium, and zinc.

Non-limiting examples of suitable metal salts include a lithium salt, asodium salt, a potassium salt, a cesium salt, a cerium salt, a magnesiumsalt, a manganese salt, an iron salt, a calcium salt, a strontium salt,a cobalt salt, a titanium salt, a aluminum salt, a copper salt, acadmium salt, and a zinc salt.

Ammonium salts can arise from the addition of ammonia or an organicamine to a compound of the invention having a carboxyl group.Non-limiting examples of suitable organic amines include triethyl amine,diisopropyl amine, ethanol amine, diethanol amine, triethanol amine,morpholine, N-methylmorpholine, piperidine, N-methylpiperidine,N-ethylpiperidine, dibenzyl amine, piperazine, pyridine, pyrrazole,imidazole, pyrazine, pipyrazine, ethylenediamine, N,N′-dibenzylethylenediamine, procaine, chloroprocaine, choline, dicyclohexyl amine, andN-methylglucamine.

Non-limiting examples of suitable ammonium salts include is atriethylammonium salt, a diisopropylammonium salt, an ethanolammoniumsalt, a diethanolammonium salt, a triethanolammonium salt, amorpholinium salt, an N-methylmorpholinium salt, a piperidinium salt, anN-methylpiperidinium salt, an N-ethylpiperidinium salt, adibenzylammonium salt, a piperazinium salt, a pyridinium salt, apyrrazolium salt, an imidazolium salt, a pyrazinium salt, anethylenediammonium salt, an N,N′-dibenzylethylenediammonium salt, aprocaine salt, a chloroprocaine salt, a choline salt, adicyclohexylammonium salt, and a N-methylglucamine salt.

Therapeutic Uses

A compound of the invention can be administered to a subject in needthereof for the treatment of a retinal degenerative disease.Non-limiting examples of retinal degenerative diseases include:retinitis pigmentosa, Leber's congenital Amaurosis, Syndromic retinaldegenerations, age-related macular degeneration including wet and dryage-related macular degeneration, and Usher Syndrome. In someembodiments, the Usher Syndrome is a subtype of Usher Syndrome. In someembodiments, the subtype is Usher I. In some embodiments, the subtype isUsher II. In some embodiments, the subtype is Usher III.

In a further embodiment of the invention, a compound of the inventioncan be administered to a subject in need thereof for the treatment ofhearing loss associated with Usher Syndrome. In some embodiments, theUsher Syndrome is a subtype of Usher Syndrome. In some embodiments, thesubtype is Usher I. In some embodiments, the subtype is Usher II. Insome embodiments, the subtype is Usher III.

A “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog,cat, horse, cow, pig, or non-human primate, such as a monkey,chimpanzee, baboon or rhesus. In one embodiment, the subject is a human.

The compounds of the invention can be administered to a subject as acomponent of a composition that comprises a pharmaceutically acceptablecarrier or vehicle. Non-limiting examples of suitable pharmaceuticalcarriers or vehicles include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesiumstearate, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol, bufferedwater, and phosphate buffered saline. These compositions can beadministered as, for example, drops, solutions, suspensions, tablets,pills, capsules, powders, and sustained-release formulations. In someembodiments, the compositions comprise, for example, lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate,and mineral oil. The compositions can additionally comprise lubricatingagents, wetting agents, emulsifying and suspending agents, preservingagents, sweetening agents or flavoring agents.

The compositions can comprise an effective amount of a compound of theinvention. An “effective amount” of a compound of the invention is anamount that is effective to treat a retinal degenerative disease orhearing loss associated with Usher Syndrome in a subject. Thecompositions can be formulated in a unit dosage form that comprises aneffective amount of a compound of the invention. In some embodiments,the compositions comprise, for example, from about 1 ng to about 1,000mg of a compound of the invention. In some embodiments, the compositionscomprise from about 100 mg to about 1,000 mg of a compound of theinvention. In some embodiments, the compositions comprise from about 100mg to about 500 mg of a compound of the invention. In some embodiments,the compositions comprise from about 200 mg to about 300 mg of acompound of the invention.

The dosage of a compound of the invention can vary depending on thesymptoms, age, and body weight of the subject, the nature and severityof the retinal degenerative disease or hearing loss associated withUsher Syndrome, the route of administration, and the form of thecomposition. The compositions described herein can be administered in asingle dose or in divided doses. In some embodiments, the dosage of acompound of the invention ranges from about 0.01 ng to about 10 g per kgbody mass of the subject, from about 1 ng to about 0.1 g per kg, or fromabout 100 ng to about 10 mg per kg.

Administration can be, for example, topical, intraaural, intraocular,parenteral, intravenous, intra-arterial, subcutaneous, intramuscular,intracranial, intraorbital, intraventricular, intracapsular,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol,suppository, or oral. Formulations for oral use include tabletscontaining a compound of the invention in a mixture with non-toxicpharmaceutically acceptable excipients. These excipients can be, forexample, inert diluents or fillers (e.g., sucrose and sorbitol),lubricating agents, glidants, and antiadhesives (e.g., magnesiumstearate, zinc stearate, stearic acid, silicas, hydrogenated vegetableoils, or talc). Formulations for ocular use can be in the form ofeyedrops.

A compound of the invention or composition thereof can be provided inlyophilized form for reconstituting, for instance, in isotonic, aqueous,or saline buffers for parental, subcutaneous, intradermal,intramuscular, or intravenous administration. A composition of theinvention can also be in the form of a liquid preparation useful fororal, intraaural, nasal, or sublingual administration, such as asuspension, syrup or elixir. A composition of the invention can also bein a form suitable for oral administration, such as a capsule, tablet,pill, and chewable solid formulation. A composition of the invention canalso be prepared as a cream for dermal administration as a liquid, aviscous liquid, a paste, or a powder. A composition of the invention canalso be prepared as a powder for pulmonary administration with orwithout an aerosolizing component.

The compositions can be in oral, intraaural, intranasal, sublingual,intraduodenal, subcutaneous, buccal, intracolonic, rectal, vaginal,mucosal, pulmonary, transdermal, intradermal, parenteral, intravenous,intramuscular and ocular dosage forms as well as being able to traversethe blood-brain barrier.

The compositions of the invention can be administered by various meansknown in the art. For example, the compositions of the invention can beadministered orally, and can be formulated as tablets, capsules,granules, powders or syrups. Alternatively, compositions of theinvention can be administered parenterally as injections (for example,intravenous, intramuscular or subcutaneous), drop infusion preparationsor suppositories. For ophthalmic application compositions of theinvention can be formulated as eye drops or eye ointments. Auralcompositions can be formulated as ear drops, ointments, creams, liquids,gels, or salves for application to the ear, either internally orsuperficially. These formulations can be prepared by conventional means,and the compositions can be mixed with any conventional additive, suchas an excipient, a binder, a disintegrating agent, a lubricant, asolubilizing agent, a suspension aid, an emulsifying agent, or a coatingagent.

Compositions of the invention can include wetting agents, emulsifiers,and lubricants, coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants.

Compositions can be suitable, for example, for oral, intraaural,intraocular, nasal, topical (including buccal and sublingual), rectal,vaginal, aerosol and/or parenteral administration. The compositions canbe provided in a unit dosage form, and can be prepared by any methodsknown in the art.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges, powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia. Compositions of the invention can also beadministered as a bolus, electuary, or paste.

Additional examples of pharmaceutically acceptable carriers or vehiclesinclude: (1) fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such ascarboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as acetyl alcohol and glycerol monostearate; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and mixtures thereof; (10) coloring agents; and (11) bufferingagents. Similar compositions can be employed as fillers in soft- orhard-filled gelatin capsules.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, gels, solutions, suspensions,syrups and elixirs. The liquid dosage form can contain inert diluentscommonly used in the art, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, diethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils such as,cottonseed, groundnut, corn, germ, olive, castor and sesame oils,glycerol, tetrahydrofuryl alcohol, polyethylene glycols, fatty acidesters of sorbitan, and mixtures thereof.

Suspension dosage forms can contain suspending, for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, and mixtures thereof.

The dosage forms for transdermal administration of a subject compositioninclude drops, powders, sprays, ointments, pastes, creams, lotions,gels, solutions, and patches. The ointments, pastes, creams, and gelscan contain excipients, such as animal and vegetable fats, oils, waxes,paraffin, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonite, silicic acid, talc and zinc oxide, ormixtures thereof.

Powders and sprays can contain excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates, polyamide powder, ormixtures thereof. Sprays may additionally contain customary propellants,such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Compositions can be administered by aerosol of solid particles. Anon-aqueous (e.g., fluorocarbon propellant) suspension could be used.Sonic nebulizers can be used because they minimize exposure to shear,which might cause degradation.

An aqueous aerosol can be made by formulating an aqueous solution orsuspension of a compound of the invention with any conventionalpharmaceutically acceptable carriers or vehicles such non-ionicsurfactants (Tweens, Pluronics, or polyethylene glycol); proteins suchas serum albumin; sorbitan esters; fatty acids; lecithin; amino acids;buffers; salts; sugars; or sugar alcohols.

Compositions suitable for parenteral administration comprise a compoundof the invention and one or more pharmaceutically acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions, oremulsions, or sterile powders which can be reconstituted into sterileinjectable solutions or dispersions just prior to use, which can containantioxidants, buffers, bacteriostats, or solutes, which render theformulation isotonic with the blood of the subject, and suspending orthickening agents.

Having described the invention with reference to certain embodiments,other embodiments will become apparent to one skilled in the art fromconsideration of the specification. The invention is further defined byreference to the following examples. It will be apparent to thoseskilled in the art that many modifications, both to materials andmethods, may be practiced without departing from the scope of theinvention.

EXAMPLES

General Synthetic Methods

Standard acidic LC-MS conditions: (10 cm_ESCI_Formic_MeCN)

A Phenomenex Luna 5 μm C18 (2), 100×4.6 mm (plus guard cartridge) columnusing an acetonitrile (Far UV grade) with 0.1% (V/V) formic acid: water(high purity via PureLab Option unit) with 0.1% formic acid gradient wasused. The flow rate was 2 mL/min. UV detection was done using a Watersdiode array detector (start Range 210 nm, end range 400 nm, rangeinterval 4 nm). Mass detection was performed via a single quadrapoleLC-MS instrument. Ionisation is either ESI or APCI dependent on compoundtypes. The gradient used ran from 95% of aqueous solvent at time 0.00min to 5% of aqueous solvent at 3.50 min. This percentage was then heldfor a further 2 min.

Standard Basic LC-MS Conditions: (10 cm_ESCI_Bicarb_MeCN):

A Waters Xterra MS 5 μm C18, 100×4.6 mm (plus guard cartridge) using anacetonitrile (Far UV grade): water (high purity via PureLab Option unit)with 10 mM ammonium bicarbonate (ammonium hydrogen carbonate) gradientwas used. The flow rate was 2 mL/min. UV detection was done using aWaters diode array detector (start Range 210 nm, end range 400 nm, rangeinterval 4 nm). Mass detection was performed via a single quadrapoleLC-MS instrument. Ionisation is either ESI or APCI dependent on compoundtypes. The gradient used ran from 95% of aqueous solvent at time 0.00min to 5% of aqueous solvent at 4.0 min. This percentage was then heldfor a further 1.5 min.

Standard Acidic HPLC Conditions: (10 cm_Formic _ACE 3 C18 AR_HPLC_CH3CN)

A Hichrom ACE 3 C18-AR mixed mode 100×4.6 mm column using anacetonitrile (Far UV grade) with 0.1% (V/V) formic acid: water (highpurity via PureLab Option unit) with 0.1% formic acid gradient was used.The flow rate was 1 mL/min. UV detection was done using an Agilent diodearray detector (300 nm, band width 200 nm; ref 450 nm, band width 100nm). The gradient used ran from 98% of aqueous solvent from time 0.00min to 3.00 min, to 100% of aqueous solvent at 12.00 min. Thispercentage was then held for a further 2.4 min.

Standard Basic HPLC conditions: (15 cm_Bicarb_GeminiNX_HPLC)

A Phenomenex, Gemini NX, 3 μm C18, 150×4.6 mm column using anacetonitrile (Far UV grade): water (high purity via PureLab Option unit)with 10 mM ammonium bicarbonate gradient was used. The flow rate was 1mL/min. UV detection was done using an Agilent diode array detector (300nm, band width 200 nm; ref 450 nm, band width 100 nm). The gradient usedran from 95.5% of aqueous solvent at time 0.00 min to 0% of aqueoussolvent at 9.00 min. This percentage was then held for a further 4.5min.

Synthetic Schemes

Non-limiting examples of synthetic schemes that are useful forsynthesizing the Pyrazolopyridazine compounds include the following.

Example 14-chloro-3-(1-methyl-1H-pyrazol-4-yl)-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Ic) Step 1: N-(1H-pyrazol-5-yl)acetamide

To a solution of 1H-pyrazol-5-amine (50 g, 0.602 mol) andN-methylmorpholine (160 mL, 1.44 mol) in CH₂Cl₂ (2 L) was added acetylchloride (99 mL, 1.38 mol) dropwise at 0° C. under an atmosphere ofnitrogen. The reaction mixture was stirred at room temperature for 1 d.Some di-acylated product was observed in an LCMS. The reaction mixturewas concentrated in vacuo and the resulting solid was suspended in MeOH(2 L) and cooled to 0° C. 4 M NaOH solution (aq., 440 mL, 1.75 mol) wasadded slowly and the mixture allowed to warm to room temperature over1.5 h. The MeOH was removed in vacuo and the solid was collected byfiltration, washed with minimal cold water and dried in vacuo to providethe title compound as a solid (60 g).

Step 2: N-(4-iodo-1H-pyrazol-5-yl)acetamide

A suspension of N-(1H-pyrazol-5-yl)acetamide (60 g, 0.48 mol), iodicacid (21.1 g, 0.12 mol) and iodine (61 g, 0.24 mol) in ethanol (1.6 L)was heated at 60° C. for 1.5 h and cooled to room temperature. Thereaction mixture was concentrated in vacuo and partitioned between ethylacetate and 2 M Na₂S₂O₃ aq. solution. The layers were separated and theaqueous extracted with ethyl acetate. The combined organic layers weredried (MgSO₄), filtered and concentrated in vacuo to provide the titlecompound as a solid (105 g).

Step 3: N-(4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide

Nitrogen was bubbled through a suspension ofN-(4-iodo-1H-pyrazol-5-yl)acetamide (30 g, 120 mmol), 10% palladium oncarbon (50% water, 7.4 g, 3 mmol), copper(I) iodide (1.14 g, 6 mmol),triphenylphosphine (6.3 g, 24 mmol) and triethylamine (50 mL, 360 mmol)in ethanol (600 mL) for 20 min. Phenyl acetylene was added and nitrogenbubbled through the mixture for a further 25 min. The reaction mixturewas then heated and stirred under reflux conditions in an atmosphere ofnitrogen for 3 d and cooled to room temperature. The reaction mixturewas filtered through celite and the filtrate was concentrated in vacuo.The residue was purified by column chromatography (silica gel,iso-hexanes/ethyl acetate 9:1 to 0:1) yielding the title compound as asolid (17.6 g).

Step 4: 1-(1-ethoxyethyl)-4-(phenylethynyl)-1H-pyrazol-5-amine

A solution of N-(4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide (17.6 g, 78mmol), ethoxyethene (11.2 mL, 117 mmol) and HCl in 1,4-dioxane (1 mL, 4mmol) in CH₂Cl₂ (520 mL) was stirred at room temperature for 1 h andconcentrated in vacuo. The residue was dissolved in ethanol (260 mL) and25% aq. NaOH solution (260 mL) and the reaction mixture was heated to75° C. for 4 h and cooled to room temperature. The ethanol was partconcentrated in vacuo and the resulting solid collected by filtration,washed with water and minimum cold ethanol and dried in vacuo to providethe title compound as a solid (16 g).

Step 5: 4-chloro-5-phenyl-1H-pyrazolo[3,4-c]pyridazine

Sodium nitrite (4.3 g, 63 mmol) was added to conc. HCl (314 mL) at −15°C. and stirred for 10 min.1-(1-ethoxyethyl)-4-(phenylethynyl)-1H-pyrazol-5-amine (3 g, 31.4 mmol)was added and the mixture stirred at −10° C. for 10 min and roomtemperature for 1 d. The reaction mixture was cooled to 0° C. and CH₂Cl₂(250 mL) was added. Under vigorous stirring, Na₂CO₃ (160 g) was addedcarefully followed by sat. aq. NaHCO₃ solution over a period of 2 huntil the pH was 7 and there was no more foaming on further addition ofbase. The layers were separated and the aqueous phase was extracted withCH₂Cl₂. The combined organic layers were dried (MgSO₄), filtered andconcentrated in vacuo. The residue was purified by column chromatography(silica gel, iso-hexanes/diethyl ether 1:0 to 0:1) yielding the titlecompound as a solid (3.43 g).

Step 6: 4-chloro-3-iodo-5-phenyl-1H-pyrazolo[3,4-c]pyridazine

A suspension of 4-chloro-5-phenyl-1H-pyrazolo[3,4-c]pyridazine (2.44 g,10.6 mmol) and N-iodosuccinimide (3.58 g, 15.9 mmol) in acetonitrile(106 mL) was heated at reflux for 1 d. The yellow solid was collected byfiltration whilst warm to provide a mixture of the title compound andstarting material (9:1, 4 g).

Step 7:4-Chloro-3-iodo-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 1a),4-chloro-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 1b) and4-chloro-3-iodo-2-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-2H-pyrazolo[3,4-c]pyridazine(Compound 20)

A solution of 4-chloro-3-iodo-5-phenyl-1H-pyrazolo[3,4-c]pyridazine and4-chloro-5-phenyl-1H-pyrazolo[3,4-c]pyridazine (9:1, 1.4 g),2-(4-methylpiperazin-1-yl)ethanol (1.13 g, 7.8 mmol), diethylazodicarboxylate (1.37 g, 7.8 mmol) and triphenyl phosphine (2.07 g, 7.9mmol) in 1,4-dioxane (26 mL) was heated to 85° C. for 1 h and thencooled to room temperature and concentrated in vacuo. The residue waspartially purified by column chromatography (silica gel, starting withiso-hexanes/ethyl acetate 1:0 to 0:1 and finishing with ethyl acetate/4M NH₃ in MeOH 1:0 to 9:1). The residue was purified by preparative HPLCto provide Compounds 1a, 1b, and 20.

4-Chloro-3-iodo-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Ia)

¹H NMR δ (ppm) (CHCl₃-d): 7.79-7.75 (2 H, m), 7.57-7.49 (3 H, m), 4.87(2 H, t), 3.01 (2 H, t), 2.61 (4 H, br s), 2.34 (4 H, br s), 2.23 (3 H,s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.67 min; m/z 483 [M+H] 97.65% purity.

4-Chloro-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Ib)

¹H NMR δ (ppm) (CHCl₃-d): 8.21 (1 H, s), 7.84-7.81 (2 H, m), 7.56-7.47(3 H, m), 4.89 (2 H, t), 3.03 (2 H, t), 2.62 (4 H, br s), 2.35 (4 H, brs), 2.23 (3 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.48 min; m/z 357 [M+H] 99.73% purity.

4-Chloro-3-iodo-2-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-2H-pyrazolo[3,4-c]pyridazine(Compound 20)

¹H NMR δ (ppm) (CHCl₃-d): 7.83 (2 H, m), 7.56-7.47 (3 H, m), 4.78 (2 H,t), 3.10 (2 H, t), 2.66 (4 H, br s), 2.47 (4 H, br s), 2.30 (3 H, s).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.07 min; m/z 483 [M+H] 94.53% purity.

Step 8:4-chloro-3-(1-methyl-1H-pyrazol-4-yl)-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 1c)

Nitrogen was bubbled through a suspension of4-chloro-3-iodo-1-(2-(4-methylpiperazin-1-ypethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(75 mg, 0.16 mmol),1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (37mg, 0.18 mmol) and K₃PO₄ (99 mg, 0.47 mmol) in DMF (1.2 mL) and water(0.4 mL) for 20 min.1,1′-Bis(diphenylphosphino)ferrocene-palladium(II)dichloridedichloromethane complex (13 mg, 0.016 mmol) was added and the tubesealed and heated using microwave irradiation to 60° C. for 30 min. Thecrude reaction mixture was filtered and purified by preparative HPLC toprovide Compound Ic(42 mg).

¹H NMR δ (ppm) (CHCl₃-d): 8.00 (1 H, s), 7.89 (1 H, s), 7.78-7.75 (2 H,m), 7.56-7.47 (3 H, m), 4.89 (2 H, t), 4.00 (3 H, s), 3.04 (2 H, t),2.70-2.58 (4 H, m), 2.44-2.30 (4 H, m), 2.24 (3 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.47 min; m/z 437 [M+H] 98.88% purity.

Example 2(3-(4-chloro-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)phenyl)(pyrrolidin-1-yl)methanone(Compound Id)

Compound Id was synthesized according to Example 1, but using(3-(pyrrolidine-1-carbonyl)phenyl)boronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.44 (1 H, t), 7.84-7.75 (3 H, m), 7.62 (1 H,dt), 7.56-7.46 (4 H, m), 4.94 (2 H, t), 3.68 (2 H, t), 3.49 (2 H, t),3.07 (2 H, t), 2.65 (4 H, br s), 2.37 (4 H, br s), 2.24 (3 H, s),2.01-1.85 (4 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 9.9min; m/z 530 [M+H] 92.56%purity.

Example 34-chloro-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-3-(2-(trifluoromethyl)pyridin-4-yl)-1H-pyrazolo[3,4-c]pyridazine(Compound Ie)

Compound Ie was synthesized according to Example 1, but using(2-(trifluoromethyl)pyridin-4-yl)boronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (CHCl₃-d): 8.88 (1 H, d), 8.19 (1 H, s), 7.98 (1 H, dd),7.81-7.77 (2 H, m), 7.57-7.51 (3 H, m), 4.99 (2 H, t), 3.12 (2 H, t),2.80 (4 H, br s), 2.62 (4 H, br s), 2.40 (3 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.85min; m/z 502 [M+H] 90.64% purity.

Example 44-chloro-3-(3,6-dihydro-2H-pyran-4-yl)-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound If)

Compound If was synthesized according to Example 1, but using2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneinstead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.68-7.64 (2 H, m), 7.45-7.36 (3 H, m),6.23-6.20 (1 H, m), 4.76 (2 H, t), 4.29 (2 H, m), 3.89 (2 H, t), 2.92 (2H, t), 2.68-2.52 (6 H, m), 2.30 (4 H, br s), 2.17 (3 H, s).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 9.06min; m/z 439 [M+H]93.3% purity.

Example 5(E)-4-chloro-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-3-styryl-1H-pyrazolo[3,4-c]pyridazine(Compound Ig)

Compound Ig was synthesized according to Example 1, but using(E)-styrylboronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (DMSO-d₆): 7.85-7.75 (5 H, m), 7.70-7.56 (4 H, m),7.52-7.45 (2 H, m), 7.44-7.38 (1 H, m), 4.92 (2 H, t), 3.01 (2 H, t),2.57-2.54 (4 H, m), 2.32-2.20 (4 H, m), 2.15(3 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 3.01min; m/z 459 [M+H] 96.98% purity.

Example 64-chloro-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-3-(6-(trifluoromethyl)pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazine(Compound Ih)

Compound Ih was synthesized according to Example 1, but using(6-(trifluoromethyl)pyridin-3-yl)boronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (DMSO-d₆): 9.26 (1 H, d), 8.60 (1 H, dd), 8.17 (1 H, d),7.82 (2 H, m), 7.67-7.58 (3 H, m), 5.01 (2 H, t), 3.04 (2 H, t),2.57-2.54 (4 H, br s), 2.76 (4 H, br s), 2.15 (3 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.86 min; m/z 502 [M+H] 97.31% purity.

Example 7(E)-4-chloro-3-(3-methoxyprop-1-en-1-yl)-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Ii)

Compound Ii was synthesized according to Example 1, but using(E)-2-(3-methoxyprop-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneinstead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (DMSO-d₆): 7.80-7.77 (2 H, m), 7.67-7.57 (3 H, m), 7.23(1 H, dt), 6.80 (1 H, dt), 4.88 (2 H, t), 4.21 (2 H, dd), 3.40 (3 H, s),2.97 (2 H, t), 2.57-2.54 ((4 H,), 2.24 (4 H, br s), 2.14 (3 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.68 min; m/z 427 [M+H] 96.83% purity.

Example 83-(benzofuran-2-yl)-4-chloro-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Ij)

Compound Ij was synthesized according to Example 1, but usingbenzofuran-2-ylboronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.83 (2 H, m), 7.72 (1 H, d), 7.64 (1 H, d),7.57 (3 H, m), 7.50 (1 H, s), 7.42 (1 H, t), 7.33 ((1 H, t), 5.02 (2 H,t), 3.12 (2 H, t), 2.68 (4 H, br s), 2.40 (4 H, br s), 2.26 ((3 H, s).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 10.39 min; m/z 473 [M+H]93.83% purity.

Example 94-chloro-3-(1-methyl-1H-pyrrol-2-yl)-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Ik)

Compound Ik was synthesized according to Example 1,but using1-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrroleinstead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.78 (2 H, m), 7.55-7.49 (3 H, m), 6.82 (1 H,t), 6.58 (1 H, dd), 6.26 (1 H, dd), 4.91 (2 H, t), 3.71 (3 H, s), 3.08(2 H, t), 2.74 (4 H, br s), 2.52 (4 H, br s), 2.34 (3 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.79 min; m/z 436 [M+H] 96.5% purity.

Example 104-chloro-3-(2,3-dihydrobenzofuran-5-yl)-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Il)

Compound Il was synthesized according to Example 1, but using(2,3-dihydrobenzofuran-5-yl)boronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.78-7.75 (2 H, m), 7.58 (1 H, s), 7.54-7.47(4 H, m), 6.90 (1 H, d), 4.91 (2 H, t), 4.66 (2 H, t), 3.30 (2 H, t),3.08 (2 H, t), 2.72 (4 H, br s), 2.50 (4 H, br s), 2.32 (3 H, s).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 9.86 min; m/z 475 [M+H]96.92% purity.

Example 114-chloro-3-(1H-indol-2-yl)-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Im)

Tert-butyl2-(4-chloro-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)-1H-indole-1-carboxylatewas synthesized according to Example 1, but using(1-(tert-butoxycarbonyl)-1H-indol-2-yl)boronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8 which was deprotected to yield Compound Im as follows.

A solution of tert-butyl2-(4-chloro-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)-1H-indole-1-carboxylate(29 mg, 0.051 mmol) in CH₂Cl₂ (3 mL) and trifluoroacetic acid (0.8 mL)was stirred at room temperature for 1d. Solid NaHCO₃ was added until nogas was evolved and sat. aq. NaHCO₃ solution and CH₂Cl₂ was added. Thelayers were separated and the aqueous was extracted with CH₂Cl₂, thecombined organics were dried (Mg50₄), filtered and concentrated invacuo. The residue was freeze dried from acetonitrile and water toprovide Compound Im as a solid.

¹H NMR δ (ppm) (CHCl₃-d): 9.14 (1 H, s), 7.79 (2 H, m), 7.70 (1 H, d),7.60-7.50 (3 H, m), 7.49-7.42 (2 H, m), 7.29 (1 H, t), 7.15 (1 H, t),4.92 (2 H, t), 3.14 (2 H, t), 2.87 (8 H, br s), 2.56 (3 H, s).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.68 min; m/z 472 [M+H] 95.85% purity.

Example 122-(4-chloro-3-(cyclopent-1-en-1-yl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(pyrrolidin-1-yl)ethanone(Compound In)

4-Chloro-3-iodo-5-phenyl-1H-pyrazolo[3,4-c]pyridazine was synthesizedaccording to Example 1 through step 6. Sodium hydride (60% in mineraloil, 32 mg, 1.75 mmol) was added to a solution of4-chloro-3-iodo-5-phenyl-1H-pyrazolo[3,4-c]pyridazine (400 mg, 1.12mmol) and 2-chloro-1-(pyrrolidin-1-yl)ethanone (54 mg, 1.8 mmol) in dryDMF (7.5 mL) at room temperature. After 1.5 h at room temperaturefurther sodium hydride (60% in mineral oil, 27 mg) was added and thesuspension stirred for 2 h. 4% LiCl aq. solution and ethyl acetate wasadded. The layers were separated and the aqueous was extracted withethyl acetate. The combined organics were dried (MgSO₄), filtered andconcentrated in vacuo. The residue was partially purified by columnchromatography (silica gel, iso-hexanes/ethyl acetate 1:0 to 0:1). Theresulting solid was dissolved in minimum CH₂Cl₂ and diethyl ether wasadded until a solid precipitated. The solid was collected by filtrationto provide2-(4-chloro-3-iodo-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(pyrrolidin-1-yl)ethanone(246 mg).

Compound In was synthesized according to Example 1 Step 8, except2-(4-chloro-3-iodo-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(pyrrolidin-1-yl)ethanonewas used instead of4-chloro-3-iodo-1-(2-(4-methylpiperazin-1-ypethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazineand 2-(cyclopent-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane wasused instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole.

¹H NMR δ (ppm) (CHCl₃-d): 7.73 (2 H, m), 7.54-7.47 (3 H, m), 6.58 (1 H,m), 5.48 (2 H, s), 3.63 (2 H, t), 3.52 (2 H, t), 2.96 (2 H, m),2.65-2.59 (2 H, m), 2.11-2.01 (4 H, m), 1.94-1.87 (2 H, m).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.52 min; m/z 408 [M+H] 97.97% purity.

Example 134-chloro-3-(5-methyl-2-furyl)-1-[2-(4-methylpiperazin-1-yl)ethyl]-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound Io)

Compound To was synthesized according to Example 1, but using(5-methyl-2-furyl)boronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.79-7.76 (2 H, m), 7.55-7.49 (3 H, m), 6.97(1 H, d), 6.17 (1 H, d), 4.92 (2 H, t), 3.05 (2 H, t), 2.64 (4 H, bs),2.44 (3 H, s), 2.36 (4 H, bs), 2.24 (3 H, s).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.87 min; m/z 437 [M+H] 96.26% purity.

Example 144-chloro-1-1-[2-[(3R)-3-fluoropyrrolidin-1-yl]ethyl]-3-iodo-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound Ip) Step 1: 2-[(3R)-3-fluoropyrrolidin-1-yl]ethanol

2-[(3R)-3-fluoropyrrolidin-1-yl]ethanol was synthesized as followed: asuspension of (3R)-3-fluoropyrrolidine hydrochloride (1 g, 8 mmol) inCH₂Cl₂ was cooled to 0° C. Triethylamine (2.79 mL, 20 mmol) and methylbromoacetate (0.83 mL, 8.8 mmol) were added and the reaction mixture wasstirred at room temperature for 16 h. The reaction mixture was dilutedwith CH₂Cl₂ and water. The layers were separated and the aqueous layerwas extracted with CH₂Cl₂. The combined organic phases were dried (phaseseparator cartridge) and concentrated in vacuo, yielding methyl2-[(3R)-3-fluoropyrrolidin-1-yl]acetate, which was used in the next stepwithout further purification (1.29 g). A solution of lithium aluminiumhydride in THF (2 M, 8 mL, 6 mmol) was slowly added to a solution ofmethyl 2-[(3R)-3-fluoropyrrolidin-1-yl]acetate (1.29 g, 8 mmol) in THF(72 mL). The reaction mixture was stirred at 70° C. for 2 h. Thereaction mixture was cooled to 0° C. and ice-cold 10% KOH aqueoussolution was added slowly. The reaction mixture was filtered and thesolid was washed with 10% KOH aqueous solution and hot ethyl acetate.The layers were separated and the aqueous was extracted with ethylacetate. The combined organics were dried (MgSO₄), filtered andconcentrated in vacuo, yielding 2-[(3R)-3-fluoropyrrolidin-1-yl]ethanolas an oil (779 mg).

Step 2:4-chloro-1-[2-[(3R)-3-fluoropyrrolidin-1-yl]ethyl]-3-iodo-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound Ip)

Compound Ip was synthesized according to Example 1, Step 7, but using2-[(3R)-3-fluoropyrrolidin-1-yl]ethanol instead of2-(4-methylpiperazin-1-yl)ethanol in Step 7. ¹H NMR δ (ppm) (CHCl₃-d):7.78-7.74 (2 H, m), 7.57-7.47 (3 H, m), 5.18-5.01 (1 H, m), 4.91-4.84 (2H, m), 3.23-3.15 (2 H, m), 2.99-2.85 (3 H, m), 2.66 (1 H, q), 2.09-1.95(2 H, m).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.65 min; m/z 472 [M+H] 94.21% purity.

Example 154-chloro-3-(3,6-dihydro-2H-pyran-4-yl)-1-[2-[(3R)-3-fluoropyrrolidin-1-yl]ethyl]-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound Iq)

Compound Iq was synthesized according to Example 1, but using2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneinstead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole and4-chloro-1-[2-[(3R)-3-fluoropyrrolidin-1-yl]ethyl]-3-iodo-5-phenyl-pyrazolo[3,4-c]pyridazineinstead of4-chloro-3-iodo-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazinein Step 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.77-7.73 (2 H, m), 7.55-7.46 (3 H, m),6.33-6.31 (1 H, m), 5.22-5.04 (1 H, m), 4.88 (2 H, t), 4.39 (2 H, q),3.99 (2 H, t), 3.20 (2 H, t), 2.98-2.89 (3 H, m), 2.76-2.63 (3 H, m),2.12-1.93 (2 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.56 min; m/z 428 [M+H]90.43% purity.

Example 164-chloro-3-iodo-5-phenyl-1-(2-pyrrolidin-1-ylethyl)pyrazolo[3,4-c]pyridazine(Compound Ir)

Compound Ir was synthesized according to Example 1 through Step 7, using2-pyrrolidin-1-ylethanol instead of 2-(4-methylpiperazin-1-yl)ethanol inStep 7.

¹H NMR δ (ppm) (CHCl₃-d): 7.79-7.75 (2 H, m), 7.57-7.49 (3 H, m), 4.89(2 H, t), 3.14 (2 H, t), 2.66-2.60 (4 H, m), 1.76-1.71 (4 H, m).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.66 min; m/z 454 [M+H] 99.44% purity.

Example 174-chloro-3-(3,6-dihydro-2H-pyran-4-yl)-5-phenyl-1-(2-pyrrolidin-1-ylethyl)pyrazolo[3,4-c]pyridazine(Compound Is)

Compound Is was synthesized according to Example 1, using2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneinstead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole and4-chloro-3-iodo-5-phenyl-1-(2-pyrrolidin-1-ylethyl)pyrazolo[3,4-c]pyridazineinstead of4-chloro-3-iodo-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazinein Step 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.76-7.73 (2 H, m), 7.55-7.46 (3 H, m),6.36-6.27 (1 H, m), 4.89 (2 H, t), 4.40-4.37 (2 H, m), 3.99 (2 H, t),3.17 (2 H, t), 2.75-2.63 (6 H, m), 1.77 (4 H, bs).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.58 min; m/z 410 [M+H] 99.45% purity.

Example 182-(4-chloro-3-iodo-5-phenyl-pyrazolo[3,4-c]pyridazin-1-yl)-1-[(3R)-3-fluoropyrrolidin-1-yl]ethanone(Compound It) Step 1: 2-chloro-1-[(3R)-3-fluoropyrrolidin-1-yl]ethanone

Chloroacetyl chloride (632 μL, 7.94 mmol) was added dropwise to asolution of (3R)-3-fluoropyrrolidine (1 g, 7.94 mmol) and triethylamine(2.2 mL, 15.9 mmol) in CH₂Cl₂ (20 mL) at 5° C. The reaction mixture wasstirred at room temperature for 1 h. Water and CH₂Cl₂ were added. Thelayers were separated and the aqueous was extracted with CH₂Cl₂. Thecombined organics were dried (MgSO₄), filtered and concentrated invacuo, to yield 2-chloro-1-[(3R)-3-fluoropyrrolidin-1-yl]ethanone (4.7g).

Step 2:2-(4-chloro-3-iodo-5-phenyl-pyrazolo[3,4-c]pyridazin-1-yl)-1-[(3R)-3-fluoropyrrolidin-1-yl]ethanone(Compound It)

Sodium hydride (60% in mineral oil, 240 mg, 6 mmol) was added to asolution of 4-chloro-3-iodo-5-phenyl-1H-pyrazolo[3,4-c]pyridazine (1.06g, 3 mmol) and 2-chloro-1-[(3R)-3-fluoropyrrolidin-1-yl]ethanone (740mg, 4.5 mmol) in dry DMF (20 mL) at room temperature. The reactionmixture was stirred at room temperature for 16 h. 4% LiCl aq. solutionand ethyl acetate was added. The layers were separated and the aqueouswas extracted with ethyl acetate. The combined organics were dried(MgSO₄), filtered and concentrated in vacuo. The residue was partiallypurified by column chromatography (silica gel, isohexane/ethyl acetate1:0 to 3:7). The resulting solid was dissolved in minimum CH₂Cl₂ anddiethyl ether was added until a solid precipitated. The solid wascollected by filtration to provide Compound It as a solid (846 mg).

¹H NMR δ (ppm) (CHCl₃-d): 7.76-7.72 (2 H, m), 7.56-7.49 (3 H, m),5.61-5.45 (2 H, m), 5.36-5.20 (1 H, m), 3.97-3.75 (3 H, m), 3.67-3.53 (1H, m), 2.55-1.90 (1 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.21 min; m/z 486 [M+H]95.11% purity.

Example 19 4-chloro-3-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Iu) Step 1: N-(3-methyl-1H-pyrazol-5-yl)acetamide

Acetyl chloride (41 g, 0.530 mol) was added dropwise to a solution of3-methyl-1H-pyrazol-5-amine (25.6 g, 0.264 mol) and N-methylmorpholine(58 mL, 0.530 mol) in CH₂Cl₂ (250 mL) at 0° C. under an atmosphere ofnitrogen. The reaction mixture was stirred at room temperature for 16 h.Water was added to the reaction mixture and the organic layerconcentrated in vacuo. The residue was taken up in a mixture of MeOH/THF(100 mL/100 mL), cooled to 10° C. and treated with 1M NaOH solution. Thereaction mixture was stirred for 0.25 h, acidified to pH 5 and organicsolvents were removed in vacuo. The resulting precipitate was filtered,washed (water, diethyl ether) and dried to provide the title compound asa white solid (29.6 g).

Step 2: N-(4-iodo-3-methyl-1H-pyrazol-5-yl)acetamide

A suspension of N-(3-methyl-1H-pyrazol-5-yl)acetamide (29.6 g, 0.213mol), iodic acid (9.3 g, 0.053 mol) and iodine (32.5 g, 0.128 mol) inethanol (300 mL) was heated at 50° C. for 3 h and cooled to roomtemperature. The reaction mixture was concentrated in vacuo and taken upin ethyl acetate. The solution was washed twice with 2 M Na₂S₂O₃followed by brine solution. The organic layer was dried (magnesiumsulphate), filtered and concentrated in vacuo. The residue wastriturated from diethyl ether, filtered and dried to provide the titlecompound as a white solid (32.5 g).

Step 3: N-(3-methyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide

Nitrogen was bubbled through a mixture ofN-(4-iodo-3-methyl-1H-pyrazol-5-yl)acetamide (32.5 g, 0.122 mol), phenylacetylene (25.0 g, 0.245 mol), triethylamine (300 mL) and DMF (100 mL)for 15 min. Copper iodide (2.3 g, 12 mmol) andbis(triphenylphosphine)palladium(II) dichloride (4.2 g, 6.0 mmol) wereadded and the reaction mixture stirred at 90° C. under nitrogen for 3 h.The reaction mixture was cooled to room temperature, diluted with ethylacetate and water. The organic phase was washed with water and brine,dried (MgSO₄), filtered and concentrated in vacuo. The residue waspurified by column chromatography (silica gel, isohexane/ethyl acetate5:1 to 1:1) yielding the title compound as a solid (15 g).

Step 4: 3-methyl-4-(phenylethynyl)-1H-pyrazol-5-amine

A mixture of N-(3-methyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide (15g, 63 mmol), ethanol (50 mL) and 25% aq. NaOH solution (50 mL) wasstirred at 90° C. for 1 h and cooled to room temperature. The reactionmixture was diluted with ethyl acetate and water. The organic phase waswashed with water and brine, dried (phase separator cartridge) andconcentrated in vacuo. Diethyl ether was added to the residue and thesolid was collected by filtration, washed with diethyl ether and driedin vacuo to provide the title compound as a solid (3.3 g).

Step 5: 4-chloro-3-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Iu)

Sodium nitrite (2.3 g, 33.8 mmol) was added portionwise to cHCl (33 mL)at −15° C. and stirred for 15 min.3-methyl-4-(phenylethynyl)-1H-pyrazol-5-amine (3.3 g, 16.9 mmol) wasadded as a solid, followed by the addition of CH₂Cl₂ (5 mL). Thereaction mixture was allowed to warm up and stirred at room temperaturefor 1 h. The reaction mixture was diluted with CH₂Cl₂ (28 mL) and NaCl(1.0 g) was added. The reaction mixture was heated to 50° C. for 16 h,then cooled to room temperature and partitioned between water andCH₂Cl₂. The organic layer was washed with water and brine, dried (phaseseparator cartridge) and concentrated in vacuo. The residue was purifiedby column chromatography (silica gel, CH₂Cl₂, then CH₂Cl₂/ethyl acetate9:1) yielding Compound Iu as a light yellow solid (1.9 g).

¹H NMR δ (ppm) (CHCl₃-d): 11.79 (1 H, s), 7.80-7.77 (2 H, m), 7.57-7.48(3 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 9.56 min; m/z 245 [M+H]95.61% purity.

Example 20 General Procedure for Mitsunobu Reaction

A mixture of 4-chloro-3-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(0.33 mmol), the alcohol (0.65 mmol), diethyl azodicarboxylate (114 mg,0.65 mmol) and triphenyl phosphine (171 mg, 0.65 mmol) in 1,4-dioxane (2mL) was heated using microwave irradiation to a temperature between 85and 120° C. for a 30 to 90 min period. The reaction mixture wasconcentrated in vacuo and the residue was purified by preparative HPLCto provide the title compound.

Example214-chloro-3-methyl-1-[2-(4-methylpiperazin-1-yl)ethyl]-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound Iv)

Compound Iv was synthesized from4-chloro-3-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine and2-(4-methylpiperazin-1-yl)ethanol following the general procedure forthe Mitsunobu reaction described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.80-7.77 (2 H, m), 7.56-7.48 (3 H, m), 4.80(2 H, t), 2.99 (2 H, t), 2.80 (3 H, s), 2.64 (4 H, br s), 2.40 (4 H, brs), 2.26 (3 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.53 min; m/z 371 [M+H] 99.25% purity.

Example 222-(4-chloro-3-methyl-5-phenyl-pyrazolo[3,4-c]pyridazin-1-yl)-1-pyrrolidin-1-yl-ethanone(Compound Iw)

Compound Iw was synthesized from4-chloro-3-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine and2-hydroxy-1-(pyrrolidin-1-yl)ethanone following the general procedurefor the Mitsunobu reaction described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.78-7.74 (2 H, m), 7.54-7.50 (3 H, m), 5.44(2 H, s), 3.63 (2 H, t), 3.52 (2 H, t), 2.81 (3 H, s), 2.07 (2 H, q),1.90 (2 H, q).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.15 min; m/z 356 [M+H] 98.98% purity.

Example 234-chloro-3-cyclopropyl-1-(2-methylsulfonylethyl)-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound Ix) Step 1:4-Chloro-3-cyclopropyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine

4-Chloro-3-cyclopropyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine wassynthesized according to Example 19, except using3-cyclopropyl-1H-pyrazol-5-amine instead of 3-methyl-1H-pyrazol-5-amine.

Step 2:4-chloro-3-cyclopropyl-1-(2-methylsulfonylethyl)-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound Ix)

Compound Ix was synthesized from4-chloro-3-cyclopropyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine and2-(methylsulfonyl)ethanol following the general procedure for theMitsunobu reaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.78-7.75 (2 H, m), 7.57-7.50 (3 H, m), 5.13(2 H, t), 3.76 (2 H, t), 2.98 (3 H, s), 2.60-2.55 (1 H, m), 1.16-1.10 (4H, m).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.37 min; m/z 377 [M+H] 99.7% purity.

Example 244-chloro-1-[2-(4-methylpiperazin-1-yl)ethyl]-5-phenyl-3-(2-pyridyl)pyrazolo[3,4-c]pyridazine(Compound Iv)

Nitrogen was bubbled through a mixture of4-chloro-3-iodo-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(100 mg, 0.21 mmol) in DMF (1.4 mL) for 10 min.Tetrakis(triphenylphosphine)palladium (24 mg, 0.021 mmol) was added andnitrogen was bubbled in the resulting mixture for another 10 min.2-(Tributylstannyl)pyridine was added and nitrogen was bubbled foranother 10 min. The tube was sealed and heated using microwaveirradiation to 100° C. for 1 h, 120° C. for 1 h, 130° C. for 2 h, thento 140° C. for 3 h. The crude reaction mixture was filtered andpartially purified by preparative HPLC. The residue was dissolved inDMSO (2 mL) and water was added. The solid was filtered off, washed withwater and dried to provide Compound Iy as a solid (10 mg).

¹H NMR δ (ppm) (CHCl₃-d): 8.81-8.77 (1 H, m), 7.90-7.78 (4 H, m),7.55-7.46 (3 H, m), 7.39 (1 H, m), 4.97 (2 H, t, J=6.7 Hz), 3.08 (2 H,t, J=6.7 Hz), 2.70-2.55 (4 H, br s), 2.36 (4 H, br s), 2.23 (3 H, s).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 8.98 min; m/z 434 [M+H]96.6% purity.

Example 254-chloro-1-1-[2-(4-methylpiperazin-1-yl)ethyl]-5-phenyl-3-pyrrolidin-1-yl-pyrazolo[3,4-c]pyridazine(Compound Iz)

Step 1:3-iodo-1-[2-(4-methylpiperazin-1-yl)ethyl]-5-phenyl-pyrazolo[3,4-c]pyridazin-4-ol

A suspension of4-chloro-3-iodo-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(140 mg, 0.29 mmol) in DMSO (3 mL) and NaOH aqueous solution (4 M, 3 mL)was heated to 50° C. for 2.5 h. The mixture was left to cool to rt, thenneutralised to pH 2-3 before loading on an SCX (log) cartridge. Thecartridge was eluted with MeOH then CH₂Cl₂/MeOH (1:1) and finally theproduct was released with 10% methanolic ammonia (7 N) in CH₂Cl₂/MeOH(1:1). Evaporation of the solvent gave the title compound (100 mg).

Step 2:1-[2-(4-methylpiperazin-1-yl)ethyl]-5-phenyl-3-pyrrolidin-1-yl-pyrazolo[3,4-c]pyridazin-4-ol

To a solution of3-iodo-1-[2-(4-methylpiperazin-1-yl)ethyl]-5-phenyl-pyrazolo[3,4-c]pyridazin-4-ol(120 mg, 0.26 mmol) in dioxane (1.7 mL) and DMSO (0.9 mL) was addedpyrrolidine (233 μL, 2.8 mmol) and sodium t-butoxide (37 mg, 0.39 mmol)and nitrogen was bubbled in the resulting mixture for 30 min. Pd₂dba₃(24 mg, 0.026 mmol) and Xantphos (9 mg, 0.016 mmol) were then added, thetube flushed with nitrogen, sealed and heated to 100° C. for 5 h.Pyrrolidine (40 μL, 0.49 mmol), Pd₂dba₃ (24 mg, 0.026 mmol) and Xantphos(9 mg, 0.016 mmol) were added and the mixture was heated to 100° C. foranother 1 h. The mixture was left to cool to rt, then neutralised to pH2-3 before loading on an SCX (10 g) cartridge. The cartridge was elutedwith MeOH then CH₂Cl₂/MeOH (1:1) and finally the product was releasedwith 10% methanolic ammonia (7 N) in CH₂Cl₂/MeOH (1:1). Evaporation ofthe solvent gave the title compound as a yellow glass (94 mg) which wasreacted as such in the next step.

Step 3:4-chloro-1-[2-(4-methylpiperazin-1-yl)ethyl]-5-phenyl-3-pyrrolidin-1-yl-pyrazolo[3,4-c]pyridazine(Compound Iz)

A suspension of1-[2-(4-methylpiperazin-1-yl)ethyl]-5-phenyl-3-pyrrolidin-1-yl-pyrazolo[3,4-c]pyridazin-4-ol(90 mg) in POCl₃ (6 mL) and CH₂Cl₂ (3 mL) was heated to 60° C. for 2.5h. The mixture was concentrated in vacuo and the residue was partitionedbetween CH₂Cl₂ and sat. aq. NaHCO₃ solution. The layers were separatedand the aqueous was extracted with CH₂Cl₂, the combined organics weredried (MgSO₄), filtered and concentrated in vacuo. The residue waspurified by preparative HPLC to provide Compound Iz (12 mg).

¹H NMR δ (ppm) (CHCl₃-d): 7.75-7.71 (2 H, m), 7.53-7.43 (3 H, m), 4.69(2 H, t, J=6.8 Hz), 3.62-3.55 (4 H, m), 2.94 (2 H, t, J=6.8 Hz), 2.63 (4H, br s), 2.40 (4 H, br s), 2.26 (3 H, s), 2.02-1.97 (4 H, m).

LCMS (15 cm_Formic_ASCENTIS_HPLC_CH3CN) Rt 7.86 min; m/z 426 [M+H] 94.3%purity.

Example 262-[4-chloro-5-phenyl-3-(trifluoromethyl)pyrazolo[3,4-c]pyridazin-1-yl]-1-pyrrolidin-1-yl-ethanone(Compound Iaa)

Step 1: Synthesis of N-[3-(trifluoromethyl)-1H-pyrazol-5-yl]acetamide

Acetyl chloride (7.7 ml, 108 mmol) was added over a period of 45 minutesto a solution of 3-(trifluoromethyl)-1H-pyrazol-5-amine (6.5 g, 43 mmol)and N-methylmorpholine (12.3 ml, 112 mmol) in CH₂Cl₂ (160 ml) withcooling in an ice bath. The reaction was allowed to warm to roomtemperature and stirred for 16 h, the solvent was removed in vacuo andthe residue dissolved in methanol (150 ml). The solution was cooled inan ice bath and 25% aqueous NaOH (7.3 ml, 65 mmol) was added. After 3.25h, 25% aqueous NaOH (0.5 ml, 4.4 mmol) was added and the reactionstirred for an additional 1.5 h. 2N HCl (20 ml) was added and theorganic solvents were removed in vacuo at below 35° C. Water was addedand the crude then extracted EtOAc (×2) and the extracts dried (MgSO₄)and concentrated in vacuo. The resultant residue was suspended in CH₂Cl₂(20 ml) with stirring. The solid was filtered and washed with CH₂Cl₂(2×3 ml) and dried in vacuo to provide the title compound as a whitesolid (7.3 g) which was used in the subsequent step.

Step 2: Synthesis ofN-[4-iodo-3-(trifluoromethyl)-1H-pyrazol-5-yl]acetamide

To a solution of N-[3-(trifluoromethyl)-1H-pyrazol-5-yl]acetamide (7.3g, 38 mmol) in ethanol (120 mL) were added iodic acid (1.65 g, 9.4 mmol)and iodine (4.8 g, 18.9 mmol). The reaction was stirred for 3.25 h at65° C. The reaction mixture was concentrated in vacuo and the residuewas suspended in hot CH₂Cl₂ (150 ml) with stirring, the solid wasfiltered and resuspended in hot CH₂Cl₂ (100 ml) filtered and dried invacuo to provide the title compound as a white solid (8.9 g) which wasused in the subsequent step.

Step 3: Synthesis ofN-[4-(2-phenylethynyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]acetamide

A degassed solution of phenylacetylene (3.9 ml, 35 mmol) and Et₃N (36mL) in DMF (13.5 mL) was added toN-[4-iodo-3-(trifluoromethyl)-1H-pyrazol-5-yl]acetamide (4.5 g, 14mmol), CuI (530 mg, 2.8 mmol) and PdCl₂(PPh₃)₂ (980 mg, 1.4 mmol) underan atmosphere of N₂. The reaction was then heated to 85° C. for 3.5 h.The reaction mixture was concentrated in vacuo and the residue waspurified by column chromatography (silica gel, ethyl acetate/isohexane1:9 to 1:0) yielding the title compound as a brown oil (3 g) which wasused in the subsequent step.

Step 4: Synthesis of4-(2-phenylethynyl)-3-(trifluoromethyl)-1H-pyrazol-5-amine

A solution ofN-[4-(2-phenylethynyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]acetamide(900 mg, 3.07 mmol) in ethanol (12 mL) and 25% NaOH (9 mL) was heated to70° C. for 1.5 h. The organic layer was separated and the aqueousextracted with EtOAc (×2). The combined organics were washed with waterand the wash extracted with EtOAc. The combined organics were dried overMgSO₄, filtered and evaporated, yielding the title compound as a red oil(652 mg) which was used in the subsequent step.

Step 5: Synthesis of4-chloro-5-phenyl-3-(trifluoromethyl)-1H-pyrazolo[3,4-c]pyridazine

To a cooled (cooling bath −15° C.) stirred suspension of sodium nitrite(540 mg, 7.8 mmol) in conc. HCl (20 mL) was added a solution of4-(2-phenylethynyl)-3-(trifluoromethyl)-1H-pyrazol-5-amine (652 mg, 2.6mmol) in CH₂Cl₂ (3 mL). The cooling bath was removed and the reactionmixture was stirred at room temperature for 1 h. NaCl (900 mg) was addedand the reaction heated at 50° C. for 16 h. CH₂Cl₂ was added to thecooled reaction mixture. The aqueous phase was extracted with CH₂Cl₂twice and the organic phases combined, dried over MgSO₄, filtered andevaporated. The residue was purified by column chromatography (silicagel, ethyl acetate/isohexane 1:9 to 1:0) yielding the title compound asa yellow glass (115 mg) which was used in the subsequent step.

Step 6: Synthesis of2-[4-chloro-5-phenyl-3-(trifluoromethyl)pyrazolo[3,4-c]pyridazin-1-yl]-1-pyrrolidin-1-yl-ethanone(Compound Iaa)

Sodium hydride (18 mg, 0.45 mmol) was added to a solution of4-chloro-5-phenyl-3-(trifluoromethyl)-1H-pyrazolo[3,4-c]pyridazine (115mg, 0.38 mmol) and 2-chloro-1-pyrrolidin-1-yl-ethanone (81 mg, 0.55mmol) in dry DMF (1.5 ml) and the reaction stirred for 21 h. Water andCH₂Cl₂ where added and the aqueous phase was extracted with CH₂Cl₂. Theorganic phases where combined, dried over MgSO₄, filtered andevaporated. The residue was purified by column chromatography (silicagel, ethyl acetate/isohexane 2:8 to 1:0) followed by preparative HPLCyielding Compound Iaa as a white solid (23 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.64 min; m/z 410 [M+H]97.54% purity.

Example 27 3-bromo-4-chloro-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Ibb)

A mixture of 4-chloro-5-phenyl-1H-pyrazolo[3,4-c]pyridazine (155 mg,0.67 mmol), bromine (51 μL, 1 mmol) and triethylamine (98 μL, 0.7 mmol)in chloroform (4 mL) was stirred at room temperature for 1 h. Additionalbromine (25 μL) and chloroform (4 mL) were added and the reactionmixture was stirred at room temperature for 4 h. Further bromine (25 μL)and triethylamine (98 μL) were added and the reaction mixture wasstirred at room temperature for 16 h. The reaction mixture was dilutedwith CH₂Cl₂ and a sodium bicarbonate solution. The aqueous phase wasextracted with CH₂Cl₂ and the combined organic phases were dried (phaseseparator cartridge) and concentrated in vacuo. The resultant residuewas purified using chromatography (silica gel, ethyl acetate/isohexane0:1 to 1:1), to provide Compound Ibb as a solid (97 mg).

¹H NMR δ (ppm)(CHCl₃-d): 11.75 (1 H, s), 7.79-7.70 (2 H, m), 7.59-7.51(3 H, m).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 11.27 min; m/z 309 [M+H]98.07% purity.

Example 284-chloro-1-[2-(4-methylpiperazin-1-yl)ethyl]-3-(5-methyl-2-thienyl)-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound Icc)

Compound Icc was synthesized following similar procedures outlined inExample 1, but using (5-methylthiophen-2-yl)boronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.67 (2 H, m), 7.43 (3 H, m), 7.36 (1 H, m),6.72 (1 H, s), 4.79 (2 H, t), 2.96 (2 H, t), 2.61 (4 H, br s), 2.50-2.38(7 H, m), 2.23 (3 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.92 min; m/z 453 [M+H] 90.98% purity.

Example 29 4-chloro-3-cyclopropyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound Idd)

Compound Idd was synthesized according to Example 19, except using3-cyclopropyl-1H-pyrazol-5-amine instead of 3-methyl-1H-pyrazol-5-amine.

¹H NMR δ (ppm) (CHCl₃-d): 10.71 (1 H, s), 7.80-7.76 (2 H, m), 7.57-7.49(3 H, m), 2.64-2.56 (1 H, m), 1.17-1.11 (4 H, m).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.76 min; m/z 271 [M+H] 98.22% purity.

Example 302-(4-chloro-3-cyclopropyl-5-phenyl-pyrazolo[3,4-c]pyridazin-1-yl)-1-pyrrolidin-1-yl-ethanone(Compound Iee)

Sodium hydride (60% in mineral oil, 18 mg, 0.44 mmol) was added to asolution of 4-chloro-3-cyclopropyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(100 mg, 0.37 mmol) and 2-chloro-1-(pyrrolidin-1-yl)ethanone (82 mg,0.55 mmol) in dry DMF (3 mL) at room temperature for 16 h. The reactionmixture was diluted with ethyl acetate and water. The organic layer waswashed with water, dried (MgSO₄), filtered and concentrated in vacuo.The residue was purified by column chromatography (silica gel,CH₂Cl₂/diethyl ether 8:2) to provide Compound Tee as a solid (65 mg).

¹H NMR δ (ppm) (CHCl₃-d): 7.76 (2 H, m), 7.56-7.45 (3 H, m), 5.39 (2 H,s), 3.65-3.57 (2 H, m), 3.54-3.45 (2 H, m), 2.61-2.53 (1 H, m),2.11-2.01 (2 H, m), 1.94-1.84 (2 H, m), 1.20-1.04 (4 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.44 min; m/z 382 [M+H]97.47% purity.

Example 312-(4-chloro-3-cyclopropyl-5-phenyl-pyrazolo[3,4-c]pyridazin-1-yl)ethanol(Compound Iff)

Compound Iff was synthesized according to Example 55, Step 4, asdescribed below, but using ethyl2-(5-amino-3-cyclopropyl-pyrazol-1-yl)acetate instead of ethyl2-(5-amino-3-phenyl-pyrazol-1-yl)acetate in Step 1.

¹H NMR δ (ppm) (CHCl₃-d): 7.79-7.75 (2 H, m), 7.57-7.49 (3 H, m), 4.79(2 H, t), 4.21-4.15 (2 H, m), 3.18 (1 H, t), 2.61-2.56 (1 H, m),1.14-1.09 (4 H, m).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 11.06 min; m/z 315 [M+H]90.2% purity.

Example 322-1-[4-chloro-5-phenyl-3-(3-pyridyl)pyrazolo[3,4-c]pyridazin-1-yl]-N-(2-dimethylaminoethyl)acetamide(Compound Igg)

Step 1: methyl 2-[5-amino-3-(3-pyridyl)pyrazol-1-yl]acetate

A mixture of 3-oxo-3-pyridin-3-ylpropane nitrile (1.31 g, 9 mmol), ethylhydrazinoacetate hydrochloride (1.39 g, 9 mmol) in methanol (9 mL) washeated under microwave irradiation at 120° C. for 1 h. The reactionmixture was concentrated in vacuo and the residue was purified by columnchromatography (silica gel, ethyl acetate) yielding the title compoundas a solid (510 mg).

Step 2: methyl 2-[5-acetamido-3-(3-pyridyl)pyrazol-1-yl]acetate

Acetic anhydride (431 μL, 4.4 mmol) was added dropwise to a solution ofmethyl 2-[5-amino-3-(3-pyridyl)pyrazol-1-yl]acetate (510 mg, 2.2 mmol)in pyridine (1.7 mL) at 0° C. under an atmosphere of nitrogen. Uponcomplete addition, the reaction mixture was warmed to room temperatureand stirred for 16 h. The reaction mixture was filtered and the solidwas washed with diethyl ether to provide the title compound as a whitesolid (430 mg).

Step 3 and 4: Methyl2-[5-acetamido-4-(2-phenylethynyl)-3-(3-pyridyl)pyrazol-1-yl]acetate

Methyl2-[5-acetamido-4-(2-phenylethynyl)-3-(3-pyridyl)pyrazol-1-yl]acetate wassynthesized according to Example 55, Steps 2 & 3, as described below,but using methyl 2-[5-acetamido-3-(3-pyridyl)pyrazol-1-yl]acetate.

Step 5: 2-[5-amino-4-(2-phenylethynyl)-3-(3-pyridyl)pyrazol-1-yl]aceticacid

To a suspension of ethyl2-[5-acetamido-4-(2-phenylethynyl)-3-(3-pyridyl)pyrazol-1-yl]acetate(410 mg, 1.1 mmol) in ethanol (10 mL) was added 25% NaOH aqueoussolution (6.5 mL). The reaction mixture was stirred at 85° C. for 6 h.The reaction mixture was cooled down to room temperature and ethanol wasevaporated in vacuo. The resulting aqueous suspension was filtered andthe solid was washed with acetonitrile and dried, yielding 235 mg of2-[5-amino-4-(2-phenylethynyl)-3-(3-pyridyl)pyrazol-1-yl]acetic acid.

Step 6:2-[4-chloro-5-phenyl-3-(3-pyridyl)pyrazolo[3,4-c]pyridazin-1-yl]aceticacid

Sodium nitrite (185 mg, 2.67 mmol) was added portionwise to cHCl (5 mL)at 0° C. and stirred for 20 min.2-[5-amino-4-(2-phenylethynyl)-3-(3-pyridyl)pyrazol-1-yl]acetic acid(285 mg, 0.89 mmol) was added as a solid. The reaction mixture wasallowed to warm up, and stirred at room temperature for 16 h. Thereaction mixture was filtered and the pH of the aqueous mother liquorwas adjusted to 2 with sodium bicarbonate and 1N HCl. The aqueous motherliquor was extracted with CH₂Cl₂ three times and the combined organicphases were dried over MgSO₄, filtered and evaporated. The residue wastriturated from diethyl ether, yielding 88 mg of a solid, consisting of2-[4-chloro-5-phenyl-3-(3-pyridyl)pyrazolo[3,4-c]pyridazin-1-yl]aceticat an estimated 35% purity by LC/MS, which was used as such in the nextstep.

Step 7:2-[4-chloro-5-phenyl-3-(3-pyridyl)pyrazolo[3,4-c]pyridazin-1-yl]-N-(2-dimethylaminoethyl)acetamide(Compound Igg)

A mixture of2-[4-chloro-5-phenyl-3-(3-pyridyl)pyrazol[3,4-c]pyridazin-1-yl]acetic(88 mg, 35% pure, 0.084 mmol), DIPEA (67 μL, 0.38 mmol), HATU (112 mg,0.30 mmol) and 2-(dimethylamino)ethylamine (32 μL, 0. 30 mmol) in CH₂Cl₂was stirred at room temperature for 1 h. The reaction mixture wasdiluted with water. The phases were separated and the aqueous phase wasextracted with CH₂Cl₂. The combined organic layer were dried (phaseseparator cartridge) and concentrated in vacuo. The residue was purifiedby preparative HPLC to provide Compound Igg. (8 mg).

¹H NMR δ (ppm) (CHCl₃-d): 9.05 (1 H, d), 8.72 (1 H, dd), 8.48 (1 H, s),8.13 (1 H, dt), 7.75-7.71 (2 H, m), 7.56-7.47 (3 H, m), 7.44 (1 H, dd),5.53 (2 H, s), 3.67-3.59 (2 H, m), 2.98 (2 H, t), 2.64 (6 H, s).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 2.79 min; m/z 436 [M+H] 95.24% purity.

Example 332-(4-chloro-3-methyl-5-phenyl-pyrazolo[3,4-c]pyridazin-1-yl)ethanol(Compound Ihh)

Compound Ihh was according to Example 55, Step 4, as described below,but using ethyl 2-(5-amino-3-methyl-pyrazol-1-yl)acetate instead ofethyl 2-(5-amino-3-phenyl-pyrazol-1-yl)acetate in Step 1.

¹H NMR δ (ppm) (CHCl₃-d): 7.78-7.75 (2 H, m), 7.56-7.49 (3 H, m),4.85-4.80 (2 H, m), 4.24-4.18 (2 H, m), 3.13-3.08 (1 H, m), 2.81 (3 H,s).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.35 min; m/z 289 [M+H] 95.23% purity.

Example 342-[4-chloro-5-phenyl-3-(3-pyridyl)pyrazolo[3,4-c]pyridazin-1-yl]ethanol(Compound Iii)

Compound Iii was synthesized according to Example 69, as shown below,but using 2-hydroxyethyl hydrazine instead of methyl hydrazine in Step3.

¹H NMR δ (ppm) (CHCl₃-d): 9.04 (1 H, s), 8.73 (1 H, m), 8.11 (1 H, m),7.76 (2 H, m), 7.54 (3 H, m), 7.45 (1 H, m), 5.01 (2 H, m), 4.31 (2 H,m), 2.92 (1 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 3.1 min; m/z 352 [M+H] 98.44% purity.

Example 354-chloro-1-[-(3-methylimidazol-4-yl)methyl]-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIa) Step 1: N-(2-acetyl-5-phenyl-pyrazol-3-yl)acetamide

To a solution of 5-phenyl-1H-pyrazol-3-amine (18.6 g, 0.117 mol) andN-methylmorpholine (30.8 mL, 0.281 mol) in CH₂Cl₂ (250 mL) was addedacetyl chloride (20 mL, 0.281 mol) dropwise at 0° C. under an atmosphereof nitrogen. The reaction mixture was stirred at room temperature for 3h. The reaction mixture was diluted with CH₂Cl₂ and water. The layerswere separated and the organic layer was washed with water and brine,dried (phase separator cartridge) and concentrated in vacuo. Diethylether was added to the residue and the solid was collected byfiltration, yielding the title compound as a solid (25.1 g).

Step 2: N-(2-acetyl-4-iodo-5-phenyl-pyrazol-3-yl)acetamide

A suspension of N-(2-acetyl-5-phenyl-pyrazol-3-yl)acetamide (25.1 g,0.103 mol), iodic acid (4.5 g, 0.026 mol) and iodine (15.7 g, 0.062 mol)in ethanol (250 mL) was heated at 50° C. for 3 h and cooled to roomtemperature. The reaction mixture was concentrated in vacuo andpartitioned between CH₂Cl₂ and 2 M Na₂S₂O₃ aq. solution. The layers wereseparated and the organic washed with brine, dried (phase separatorcartridge), and concentrated in vacuo to provide a mixture of the titlecompound and starting material (2.2:1, 30.3 g). The mixture was put inreaction again using iodic acid (1.6 g, 9.6 mmol) and iodine (9.7 g,0.038 mol) in ethanol (250 mL) under the same conditions, to provide thetitle compound as a solid (31.9 g).

Step 3: N-[3-phenyl-4-(2-phenylethynyl)-1H-pyrazol-5-yl]acetamide

Nitrogen was bubbled through a mixture ofN-(2-acetyl-4-iodo-5-phenyl-pyrazol-3-yl)acetamide (31.87 g, 86.4 mmol),phenyl acetylene (17.6 g, 173 mmol), triethylamine (200 mL) and DMF (100mL) for 15 min. Copper iodide (1.64 g, 8.6 mmol) andbis(triphenylphosphine)palladium(II) dichloride (3.0 g, 4.3 mmol) wereadded and the reaction mixture was stirred at 90° C. under nitrogen for3 h. The reaction mixture was cooled to room temperature, diluted withethyl acetate and water. The organic phase was washed with water andbrine, dried (MgSO₄), filtered and concentrated in vacuo. The residuewas purified by column chromatography (silica gel, iso-hexanes/ethylacetate 5:1 to 1:1) yielding the title compound as a solid (12.5 g).

Step 4: 3-phenyl-4-(2-phenylethynyl)-1H-pyrazol-5-amine

A mixture of N-[3-phenyl-4-(2-phenylethynyl)-1H-pyrazol-5-yl]acetamide(12.5 g, 42 mmol), ethanol (100 mL) and 25% aq. NaOH solution (100 mL)was stirred and heated to 90° C. for 1 h and cooled to room temperature.The reaction mixture was diluted with ethyl acetate and water. Theorganic phase was washed with water and brine, dried (phase separatorcartridge) and concentrated in vacuo. Diethyl ether was added to theresidue and the solid was collected by filtration, washed with diethylether and dried in vacuo to provide the title compound as a solid (5.4g).

Step 5: 4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine

Sodium nitrite (2.88 g, 42 mmol) was added portionwise to cHCl (314 mL)at −15° C. and stirred for 15 min.3-phenyl-4-(2-phenylethynyl)-1H-pyrazol-5-amine (5.4 g, 21 mmol) wasadded as a solid, followed by the addition of CH₂Cl₂ (10 mL). Thereaction mixture was allowed to warm up and stirred at room temperaturefor 1 h. The reaction mixture was diluted with CH₂Cl₂ (44 mL) and NaCl(2.7 g) was added. The reaction mixture was heated to 50° C. for 1 d.The layers were separated and the organic layer was washed with water,dried (phase separator cartridge) and concentrated in vacuo. The residuewas purified by column chromatography (silica gel, iso-hexanes/ethylacetate 4:1, then CH₂Cl₂/ethyl acetate 1:0 to 4:1) yielding the titlecompound as a solid (3.0 g).

Step 6:4-chloro-1-[(3-methylimidazol-4-yl)methyl]-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIa)

A mixture of 4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (100 mg,0.33 mmol), 1-methyl-1H-imidazol-5-yl)methanol (73 mg, 0.65 mmol),diethyl azodicarboxylate (114 mg, 0.65 mmol) and triphenyl phosphine(171 mg, 0.65 mmol) in 1,4-dioxane (2 mL) was heated using microwaveirradiation to 100° C. for 30 min. The reaction mixture was concentratedin vacuo and the residue was purified by preparative HPLC to provideCompound IIa (46 mg).

¹H NMR δ (ppm) (CHCl₃-d): 7.77-7.70 (4 H, m), 7.56-7.46 (7 H, m), 7.37(1 H, s), 5.97 (2 H, s), 3.91 (3 H, s).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.88 min; m/z 401 [M+H] 94.62% purity.

Example 36 General Procedure for Mitsunobu Reaction:

A mixture of 4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (0.33mmol), the alcohol (0.65 mmol), diethyl azodicarboxylate (114 mg, 0.65mmol) and triphenyl phosphine (171 mg, 0.65 mmol) in 1,4-dioxane (2 mL)was heated using microwave irradiation to a temperature between 85 and120° C. for a 30 to 90 min period. The reaction mixture was concentratedin vacuo and the residue was purified by preparative HPLC to provide thetitle compound.

Example 371-[2-(4-chloro-3,5-diphenyl-pyrazolo[3,4-c]pyridazin-1-yl)ethyl]pyrrolidin-2-one(Compound IIb)

Compound IIb was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine and1-(2-hydroxyethyl)pyrrolidin-2-one following the general procedure forthe Mitsunobu reaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.78-7.73 (4 H, m), 7.56-7.48 (6 H, m), 4.98(2 H, t), 3.95 (2 H, t), 3.49 (2 H, t), 2.16 (2 H, t), 2.01-1.90 (2 H,m).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.16 min; m/z 418 [M+H] 99.69% purity.

Example 384-chloro-1-(2-imidazol-1-ylethyl)-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIc)

Compound IIc was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine and2-(1H-imidazol-1-yl)ethanol following the general procedure for theMitsunobu reaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.76-7.71 (4 H, m), 7.55-7.48 (6 H, m), 7.31(1 H, s), 6.98 (1 H, d), 6.96 (1 H, d), 5.16 (2 H, t), 4.72 (2 H, t).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.38 min; m/z 401 [M+H] 98.08% pu

Example 394-chloro-3,5-diphenyl-1-(3,3,3-trifluoropropyl)pyrazolo[3,4-c]pyridazine(Compound IId)

Compound IId was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine and3,3,3-trifluoropropan-1-ol following the general procedure for theMitsunobu reaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.79-7.74 (4 H, m), 7.56-7.47 (6 H, m), 5.09(2 H, t), 3.07-2.94 (2 H, m).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 13.00 min; m/z 403 [M+H]97.24% purity.

Example 404-chloro-3,5-diphenyl-1-tetrahydropyran-4-yl-pyrazolo[3,4-c]pyridazine(Compound IIe)

Compound IIe was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine andtetrahydro-2H-pyran-4-ol following the general procedure for theMitsunobu reaction.

¹H NMR δ (ppm) (CHCl₃-d): 7.80-7.75 (4 H, m), 7.56-7.46 (6 H, m),5.52-5.46 (1 H, m), 4.23-4.19 (2 H, m), 3.71 (2 H, td), 2.60-2.54 (2 H,m), 2.18-2.14 (2 H, m).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 13.06 min; m/z 391 [M+H]96.12% purity.

Example 414-chloro-1-[(3-methyloxetan-3-yl)methyl]-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIf)

Compound IIf was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine and(3-methyloxetan-3-yl)methanol following the general procedure for theMitsunobu reaction.

¹H NMR δ (ppm) (CHCl₃-d): 7.79-7.74 (4 H, m), 7.56-7.48 (6 H, m), 5.02(2 H, s), 4.95 (2 H, d), 4.50 (2 H, d), 1.40 (3 H, s).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 11.19 min; m/z 391 [M+H]94.86% purity.

Example 424-chloro-1-[(1-methylpyrazol-4-yl)methyl]-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIg)

Compound IIg was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine and(1-methyl-1H-pyrazol-4-yl)methanol following the general procedure forthe Mitsunobu reaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.78-7.73 (4 H, m), 7.67 (1 H, s), 7.64 (1 H,s), 7.55-7.46 (6 H, m), 5.86 (2 H, s), 3.85 (3 H, s).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 12.26 min; m/z 401 [M+H]95.22% purity.

Example 434-[(4-chloro-3,5-diphenyl-pyrazolo[3,4-c]pyridazin-1-yl)methyl]oxazole(Compound IIh)

Compound IIh was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine andoxazol-4-ylmethanol following the general procedure for the Mitsunobureaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.85 (1 H, s), 7.83 (1 H, d, J=1.12 Hz),7.79-7.75 (4 H, m), 7.56-7.46 (6 H, m), 5.97 (2 H, s).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.81 min; m/z 388 [M+H]93.58% purity.

Example 444-chloro-1-(cyclopropylmethyl)-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIi)

Compound IIi was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine andcyclopropylmethanol following the general procedure for the Mitsunobureaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.81-7.76 (4 H, m), 7.55-7.45 (6 H, m), 4.69(2 H, d), 1.62-1.58 (1 H, m), 0.65-0.61 (4 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 11.77 min; m/z 361 [M+H]96.25% purity.

Example 454-chloro-3,5-diphenyl-1-[[(2R)-tetrahydrofuran-2-yl]methyl]pyrazolo[3,4-c]pyridazine(Compound IIj)

Compound IIj was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine andR)-(tetrahydrofuran-2-yl)methanol following the general procedure forthe Mitsunobu reaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.80-7.76 (4 H, m), 7.55-7.44 (6 H, m), 4.97(1 H, dd), 4.78-4.64 (2 H, m), 3.99 (1 H, q), 3.83-3.74 (1 H, m),2.16-2.03 (1 H, m), 2.03-1.80 (3 H, m).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 12.93 min; m/z 391 [M+H]92.23% purity.

Example 464-chloro-3,5-diphenyl-1(2,2,2-trifluoroethyl)pyrazolo[3,4-c]pyridazine(Compound IIk)

Compound IIk was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine and2,2,2-trifluoroethanol following the general procedure for the Mitsunobureaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.81-7.75 (4 H, m), 7.58-7.50 (6 H, m), 5.40(2 H, q).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 12.89 min; m/z 389 [M+H]97.09% purity.

Example 474-chloro-1-(2-fluoroethyl)-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIl)

Compound III was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine and 2-fluoroethanolfollowing the general procedure for the Mitsunobu reaction as describedabove.

¹H NMR δ (ppm)(CHCl₃-d): 7.80-7.75 (4 H, m), 7.56-7.49 (6 H, m),5.18-5.07 (3 H, m), 5.01 (1 H, t).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 11.05 min; m/z 353 [M+H]96.03% purity.

Example 484-(4-chloro-3,5-diphenyl-pyrazolo[3,4-c]pyridazin-1-yl)butan-2-one(Compound IIm)

Compound IIm was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine and4-hydroxybutan-2-one following the general procedure for the Mitsunobureaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.78-7.73 (4 H, m), 7.56-7.45 (6 H, m), 5.09(2 H, t), 3.34 (2 H, t), 2.26 (3 H, s).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 12.5 min; m/z 377 [M+H]95.07% purity.

Example 494-chloro-3,5-diphenyl-1-(3-pyridylmethyl)pyrazolo[3,4-c]pyridazine(Compound IIn)

Compound IIn was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine andpyridin-3-ylmethanol following the general procedure for the Mitsunobureaction.

¹H NMR δ (ppm) (CHCl₃-d): 8.85 (1 H, d), 8.57 (1 H, dd), 7.93 (1 H, dt),7.79-7.73 (4 H, m), 7.56-7.48 (6 H, m), 7.31-7.27 (1 H, m), 6.00 (2 H,s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.03 min; m/z 398 [M+H] 97.31% purity.

Example 504-chloro-3,5-diphenyl-1-(4-pyridylmethyl)pyrazolo[3,4-c]pyridazine(Compound IIo)

Compound IIo was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine andpyridin-4-ylmethanol following the general procedure for the Mitsunobureaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 8.60 (2 H, dd), 7.79-7.75 (4 H, m), 7.56-7.48(6 H, m), 7.39 (2 H, d), 5.98 (2 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 3.77 min; m/z 398 [M+H] 97.2% purity.

Example 514-chloro-3,5-diphenyl-1-(2-pyridylmethyl)pyrazolo[3,4-c]pyridazine(Compound IIp)

Compound IIp was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine andpyridin-2-ylmethanol following the general procedure for the Mitsunobureaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 8.60-8.57 (1 H, m), 7.80-7.75 (4 H, m),7.68-7.62 (1 H, m), 7.55-7.46 (7 H, m), 7.23-7.20 (1 H, m) 6.15 (2 H,s).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 11.05 min; m/z 398 [M+H]95.92% purity.

Example 524-chloro-1-(2-methylsulfonylethyl)-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIq)

Compound IIq was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine and2-(methylsulfonyl)ethanol following the general procedure for theMitsunobu reaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.78-7.74 (4 H, m), 7.56-7.48 (6 H, m),5.34-5.29 (2 H, m), 3.88 (2 H, t), 3.05 (3 H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.13 min; m/z 413 [M+H] 97.31% purity.

Example 531-[2-[4-chloro-3-(3-fluorophenyl)-5-phenyl-pyrazolo[3,4-c]pyridazin-1-yl]ethyl]pyrrolidin-2-one(Compound IIr) Step 1: N-[3-(3-fluorophenyl)-1H-pyrazol-5-yl]acetamide

To a solution of 3-(3-fluorophenyl)-1H-pyrazol-5-amine (6.5 g, 36 mmol)and N-methylmorpholine (9.7 mL, 88 mmol) in CH₂Cl₂ (150 mL) was addedacetyl chloride (6 mL, 85 mmol) dropwise at 0° C. under an atmosphere ofnitrogen. The reaction mixture was stirred at room temperature for 1 d.The reaction mixture was concentrated in vacuo. MeOH (50 mL) and THF (50mL) were added to the residue, followed by the addition of NaOH solution(aq. 2.5 M, 42.5 mL) at 0° C. The reaction mixture was stirred at roomtemperature for 15 min and HCl solution was added until pH reached ˜6.The organic solvents were evaporated in vacuo. The solid from theresulting aqueous suspension was collected by filtration, yielding thetitle compound as a solid (7.6 g).

Step 2: N-[3-(3-fluorophenyl)-4-iodo-1H-pyrazol-5-yl]acetamide

A suspension of N-[3-(3-fluorophenyl)-1H-pyrazol-5-yl]acetamide (7.6 g,34.7 mmol), iodic acid (1.5 g, 8.5 mmol) and iodine (4.4 g, 17.3 mmol)in ethanol (200 mL) was heated at 60° C. for 1 h and cooled to roomtemperature. The reaction mixture was concentrated in vacuo andpartitioned between CH₂Cl₂ and 2 M Na₂S₂O₃ aq. solution. The layers wereseparated and the organic washed with brine, dried (MgSO₄), andconcentrated in vacuo to provide the title compound as a solid (10.8 g).

Step 3:N-[3-(3-fluorophenyl)-4-(2-phenylethynyl)-1H-pyrazol-5-yl]acetamide

Nitrogen was bubbled through a mixture ofN-[3-(3-fluorophenyl)-4-iodo-1H-pyrazol-5-yl]acetamide (10.8 g, 44mmol), phenyl acetylene (12.5 g, 123 mmol), triethylamine (100 mL) andDMF (40 mL) for 15 min. Copper iodide (840 mg, 4.42 mmol) andbis(triphenylphosphine)palladium(II) dichloride (1.5 g, 2.1 mmol) wereadded and the reaction mixture was stirred at 90° C. under nitrogen for6 h. The reaction mixture was cooled to room temperature, diluted withethyl acetate and water. The organic phase was washed with water andbrine, dried (MgSO₄), filtered and concentrated in vacuo. The residuewas purified by column chromatography (silica gel, isohexane/ethylacetate 1:0 to 0:1) yielding the title compound as a solid (4 g).

Step 4: 3-(3-fluorophenyl)-4-(2-phenylethynyl)-1H-pyrazol-5-amine

A mixture ofN-[3-(3-fluorophenyl)-4-(2-phenylethynyl)-1H-pyrazol-5-yl]acetamide (2g, 6.2 mmol), ethanol (22 mL) and 25% aq. NaOH solution (22 mL) wasstirred and heated to 80° C. for 1 h and cooled to room temperature. Thereaction mixture was diluted with ethyl acetate and water. The organicphase was washed with water and brine, dried (phase separator cartridge)and concentrated in vacuo, to provide the title compound as a solid (1.2g).

Step 5:4-chloro-3-(3-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine

Sodium nitrite (740 mg, 10.7 mmol) was added portionwise to cHCl (24 mL)at −15° C. and stirred for 15 min.3-(3-fluorophenyl)-4-(2-phenylethynyl)-1H-pyrazol-5-amine (1 g, 3.6mmol) was added as a solid, followed by the addition of CH₂Cl₂ (10 mL).The reaction mixture was allowed to warm up and stirred at roomtemperature for 1.5 h. The reaction mixture was diluted with CH₂Cl₂ (20mL) and NaCl (0.5 g) was added. The reaction mixture was heated to 50°C. for 1 d. The layers were separated and the organic layer was washedwith water, dried (phase separator cartridge) and concentrated in vacuo.The residue was purified by column chromatography (silica gel, ethylacetate/isohexane 0:1 to 7:3) yielding the title compound as a solid(500 mg).

Step 6:1-[2-[4-chloro-3-(3-fluorophenyl)-5-phenyl-pyrazolo[3,4-c]pyridazin-1-yl]ethyl]pyrrolidin-2-one(Compound IIr)

Compound IIr was synthesized from4-chloro-3-(3-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine and1-(2-hydroxyethyl)pyrrolidin-2-one following the general procedure forthe Mitsunobu reaction as described.

¹H NMR δ (ppm) (CHCl₃-d): 7.77-7.74 (2 H, m), 7.59-7.51 (4 H, m),7.51-7.43 (2 H, m), 7.22-7.16 (1 H, m), 4.98 (2 H, t), 3.94 (2 H, t),3.51 (2 H, t), 2.14 (2 H, t), 2.02-1.92 (2 H, m).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.92 min; m/z 436 [M+H] 99.49% purity.

Example 544-chloro-3-(3-fluorophenyl)-1-(2-imidazol-1-ylethyl)-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound IIs)

Compound IIs was synthesized from4-chloro-3-(3-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine and2-(1H-imidazol-1-yl)ethanol following the general procedure for theMitsunobu reaction as described above.

¹H NMR δ (ppm) (CHCl₃-d): 7.77-7.73 (2 H, m), 7.55-7.46 (6 H, m), 7.36(1 H, s), 7.31-7.23 (1 H, m), 7.01-6.99 (1 H, m), 6.97 (1 H, s), 5.17 (2H, t,), 4.72 (2 H, t).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.85 min; m/z 419 [M+H] 92.97% purity.

Example 554-chloro-1-[2-[(3S)-3-fluoropyrrolidin-1-yl]ethyl]-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIt)

Step 1: Ethyl 2-(5-acetamido-3-phenyl-pyrazol-1-yl)acetate

Acetic anhydride (12.7 mL, 134.8 mmol) was added dropwise to a solutionof ethyl 2-(5-amino-3-phenyl-pyrazol-1-yl)acetate (31.5 g, 128.4 mmol)in pyridine (200 mL) at 0° C. under an atmosphere of nitrogen. Uponcomplete addition, the reaction mixture was warmed to room temperatureand stirred for 16 h. The reaction mixture was concentrated in vacuo.The residue was diluted in CH₂Cl₂ (250 mL). The organic phase was washedwith water and brine, dried (MgSO₄), filtered and concentrated in vacuo.The residue was triturated from diethyl ether, filtered and dried toprovide the title compound as a white solid (34.81 g).

Step 2: Ethyl 2-(5-acetamido-4-iodo-3-phenyl-pyrazol-1-yl)acetate

A suspension of ethyl 2-(5-acetamido-3-phenyl-pyrazol-1-yl)acetate(34.81 g, 60.6 mmol), iodic acid (5.33 g, 30.3 mmol) and iodine (15.37g, 60.6 mmol) in ethanol (250 mL) was heated at 50° C. for 1.5 h andcooled to room temperature. The reaction mixture was concentrated invacuo and the residue was dissolved in CH₂Cl₂ (250 mL). The solution waswashed twice with 2 M Na₂S₂O₃ followed by brine solution. The organiclayer was dried (magnesium sulphate), filtered and concentrated invacuo. The residue was triturated from diethyl ether, filtered and driedto provide the title compound as a solid (44.13 g).

Step 3: Ethyl2-[5-acetamido-3-phenyl-4-(2-phenylethynyl)pyrazol-1-yl]acetate

Nitrogen was bubbled through a mixture of ethyl2-(5-acetamido-4-iodo-3-phenyl-pyrazol-1-yl)acetate (34.95 g, 84.58mmol), phenyl acetylene (18.6 mL, 169.16 mmol), triethylamine (300 mL)and DMF (120 mL) for 1.5 h. Copper iodide (1.61 g, 8.46 mmol) andbis(triphenylphosphine)palladium(II) dichloride (3 g, 4.23 mmol) wereadded and the reaction mixture was stirred at 90° C. under nitrogen for2 h. The reaction mixture was concentrated in vacuo, and the residue wasco-evaporated with toluene to remove excess DMF. The residue was dilutedwith ethyl acetate (300 mL) and washed with water (2 ×100 mL). Theorganic phase was filtered through celite, washed with water and brine,dried (MgSO₄), filtered and concentrated in vacuo. The residue wastriturated from ethyl acetate, yielding the title compound as a solid(25.67 g).

Step 4: 2-[5-Amino-3-phenyl-4-(2-phenylethynyl)pyrazol-1-yl]ethanol

To a suspension of ethyl2-[5-acetamido-3-phenyl-4-(2-phenylethynyl)pyrazol-1-yl]acetate (22.4 g,58 mmol) in ethanol (290 mL) was added sodium borohydride (11 g, 289mmol) and the reaction stirred at room temperature for 16 h. Thereaction mixture was partially concentrated to a final volume of 250 mL.25% NaOH (250 mL) was added and the reaction mixture was stirred at 80°C. for 4 h. The reaction mixture was cooled down to room temperature andthe two phases were separated. The aqueous phase was extracted withethyl acetate three times and the organic phases combined, dried overMgSO₄, filtered and evaporated. The residue was triturated from diethylether (20 mL) and the product was filtered and dried in vacuo to providethe title compound as an off-white solid (9.96 g). The mother liquor wasconcentrated in vacuo and purified by column chromatography (silica gel,gradient 0 to 100% ethyl acetate/isohexane) yielding a further 1.79 g ofthe title compound.

Step 5: 2-(4-Chloro-3,5-diphenyl-pyrazolo[3,4-c]pyridazin-1-yl)ethanol

Sodium nitrite (3.42 g, 49.5 mmol) was added portionwise to cHCl (165mL) at −10° C. and stirred for 20 min.2[5-amino-3-phenyl-4-(2-phenylethynyl)pyrazol-1-yl]ethanol (5 g, 16.5mmol) was added as a solid. The reaction mixture was allowed to warm up,sonicated for 5 min then stirred at room temperature for 2 h. Thereaction mixture was diluted with CH₂Cl₂ and water and the aqueous phasewas extracted with CH₂Cl₂. The organic phases where combined, dried overMgSO₄, filtered and evaporated. The residue was partially purified bycolumn chromatography (silica gel, gradient 0 to 100% ethylacetate/isohexane). The resulting residue was then triturated fromdiethyl ether, yielding the title compound as a solid (956 mg).

Step 6:4-Chloro-1-(2-iodoethyl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine

Triphenylphosphine (160 mg, 0.62 mmol), imidazole (42 mg, 0.62 mmol) andiodine (160 mg, 0.62 mmol) were added to a solution of2-(4-chloro-3,5-diphenyl-1H pyrazolo[3,4-c]pyridazin-1-yl)ethanol (181mg, 0.52 mmol) in CH₂Cl₂ (6 mL). After stirring at ambient temperaturefor 1 h the reaction was filtered and solvent removed in vacuo.Purification using chromatography (silica gel, gradient 10 to 60% ethylacetate/ isohexane) gave4-chloro-1-(2-iodoethyl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine as aclear oil (202 mg) which was used as such in the subsequent step.

Step 7:4-Chloro-1-[2-[(3,5)-3-fluoropyrrolidin-1-y]ethyl]-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIt)

A solution of4-chloro-1-(2-iodoethyl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (90mg, 0.2 mmol) in dry CH₂Cl₂ (2 mL) was added to (S)-3-fluoropyrrolidinehydrochloride (126 mg, 1 mmol), DIPEA (0.21 mL, 1.2 mmol) was added andthe reaction stirred for 2 days. Further CH₂Cl₂ (3 mL),(S)-3-fluoropyrrolidine hydrochloride (126 mg, 1 mmol) and DIPEA (0.21mL, 1.2 mmol) was added and the reaction stirred for an additional 7days. The resultant residue was purified using chromatography (silicagel, gradient 20 to 100% ethyl acetate/isohexane), followed bypreparative HPLC to provide Compound IIt as a white solid (25 mg).

¹H NMR δ (ppm) (DMSO-d₆): 7.76-7.70 (2 H, m), 7.70-7.66 (2 H, m),7.53-7.43 (6H, m), 5.15-5.01 (1 H, m), 4.83 (2 H, t), 3.07 (2 H, t),2.93-2.77 (2 H, m), 2.74-2.58 (1 H, m), 2.41-2.45 (1 H, m), 2.07-1.90 (1H, m), 1.82-1.68 (1 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 11.27 min; m/z 422 [M+H]98.15% purity.

Example 564-chloro-1-[2-1[(3R)-3-fluoropyrrolidin-1-yl]ethyl]-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIu)

Compound IIu was synthesized according to Example 55, but using(R)-3-fluoropyrrolidine hydrochloride instead of (S)-3-fluoropyrrolidinehydrochloride.

¹H NMR δ (ppm) (DMSO-d₆): 7.76-7.70 (2 H, m), 7.70-7.66 (2 H, m),7.53-7.43 (6H, m), 5.15-5.01 (1 H, m), 4.83 (2 H, t), 3.07 (2 H, t),2.93-2.77 (2 H, m), 2.74-2.58 (1 H, m), 2.41-2.45 (1 H, m), 2.07-1.90 (1H, m), 1.82-1.68 (1 H, m).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.84 min; m/z 422 [M+H] 98.06% purity.

Example 574-chloro-1-[2-(3,3-difluoropyrrolidin-1-yl)ethyl]-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIv)

A solution of4-chloro-1-(2-iodoethyl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (91mg, 0.2 mmol) in dry CH₂Cl₂ (2 mL) was added to 3,3-difluoropyrrolidinehydrochloride (143 mg, 1 mmol), DIPEA (0.2 mL, 1.1 mmol) was added andthe reaction stirred for 5 days. The resultant residue was purifiedusing chromatography (silica gel, gradient 20 to 50% ethylacetate/isohexane, followed by preparative HPLC to provide Compound IIvas a white solid (24.5 mg).

¹H NMR δ (ppm)(DMSO-d₆): 7.74-7.70 (2 H, m), 7.70-7.65 (2 H, m),7.52-7.41 (6H, m), 4.84 (2 H, t), 3.07 (2 H, t), 2.97 (2 H, t), 2.75 (2H, t), 2.16-2.03 (2 H, m).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.45 min; m/z 440 [M+H] 99.4% purity.

Example 584-chloro-1-[2-(3,3-dilluoroazetidin-1-yl)ethyl]-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound IIw)

Compound IIw was synthesized according to Example 57, but using3,3-difluoroazetidine hydrochloride instead of 3, 3-difluoropyrrolidinehydrochloride.

¹H NMR δ (ppm)(DMSO-d₆): 7.74-7.70 (2 H, m), 7.70-7.66 (2 H, m),7.53-7.43 (6H, m), 4.74 (2 H, t), 3.56 (4 H, t), 3.15 (2 H, t).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.35 min; m/z 426 [M+H] 95.4% purity.

Example 594-chloro-3-(3-fluorophenyl)-1-[2-[(3R)-3-fluoropyrrolidin-1-yl]ethyl]-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound IIx)

Compound IIx was synthesized according to Example 1, using3-fluorophenylboronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole and4-chloro-1-[2-[(3R)-3-fluoropyrrolidin-1-yl]ethyl]-3-iodo-5-phenyl-pyrazolo[3,4-c]pyridazineinstead of4-chloro-3-iodo-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazinein Step 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.79-7.75 (2 H, m), 7.60-7.44 (6 H, m), 7.18(1 H, tdd), 5.22-5.04 (1 H, m), 4.96 (2 H, t), 3.25 (2 H, t), 3.01-2.91(3 H, m), 2.68 (1 H, q), 2.13-1.96 (2 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 11.32 min; m/z 440 [M+H]96.24% purity.

Example 604-chloro-3-(4-fluorophenyl)-1-[2-[(3R)-3-fluoropyrrolidin-1-yl]ethyl]-5phenyl-pyrazolo[3,4-c]pyridazine (Compound IIv)

Compound IIy was synthesized according to Example 1, but using4-fluorophenylboronic acid instead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole and4-chloro-1-[2-[(3R)-3-fluoropyrrolidin-1-yl]ethyl]-3-iodo-5-phenyl-pyrazolo[3,4-c]pyridazineinstead of4-chloro-3-iodo-1-(2-(4-methylpiperazin-1-yl)ethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazinein Step 8.

¹H NMR δ (ppm)(CHCl₃-d): 7.78-7.73 (4 H, m), 7.55-7.46 (3 H, m),7.23-7.15 (2H, m), 5.21-5.03 (1 H, m), 4.95 (2 H, t), 3.24 (2 H, t),3.01-2.91 (3 H, m), 2.68 (1 H, q), 2.12-1.96 (2 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 11.29 min; m/z 440 [M+H]93.55% purity.

Example 61 4-chloro-5-iodo-1-methyl-3-phenyl-pyrazolo[3,4-c]pyridazine(Compound IIIa) Step 1:N-(1-methyl-3-phenyl-4-((trimethylsilyl)ethynyl)-1H-pyrazol-5-yl)acetamide

Nitrogen was bubbled through a mixture ofN-(4-iodo-1-methyl-3-phenyl-1H-pyrazol-5-yl)acetamide (5 g, 15 mmol) inDMF (15 mL) and triethylamine (35 mL) for 15 min. Copper iodide (0.56 g,3.0 mmol), bis(triphenylphosphine)palladium(II) dichloride (0.53 g, 0.75mmol) and ethynyltrimethylsilane (3.0 g, 30 mmol) were added and thereaction mixture was stirred in a sealed tube at 90° C. under nitrogenfor 3 h. The reaction mixture was cooled to room temperature, dilutedwith ethyl acetate and water. The organic phase was washed with waterand brine, dried (phase separation cartridge) and concentrated in vacuo.The residue was purified by column chromatography (silica gel,isohexane/ethyl acetate 5:1 to 10:3) yielding the title compound as asolid (2.3 g).

Step 2: 1-(5-amino-1-methyl-3-phenyl-pyrazol-4-yl)ethanone

A mixture ofN-(1-methyl-3-phenyl-4-((trimethylsilyl)ethynyl)-1H-pyrazol-5-yl)acetamide(2.3 g, 7.4 mmol), ethanol (20 mL) and 25% aq. NaOH solution (20 mL) washeated with stirring at 90° C. for 6 h and cooled to room temperature.The reaction mixture was diluted with ethyl acetate and water. Theorganic phase was washed with water and brine, dried (phase separatorcartridge) and concentrated in vacuo. The residue was purified usingchromatography (silica gel, gradient 0 to 40% ethyl acetate/CH₂Cl₂),followed by trituration in diethyl ether to provide the title compoundas a white solid (724 mg).

Step 3: 1-methyl-3-phenyl-pyrazolo[3,4-c]pyridazin-4-ol

A suspension of 1-(5-amino-1-methyl-3-phenyl-pyrazol-4-yl)ethanone (241mg, 1.12 mmol) in cHCl (6.7 mL) and water (1 mL) was cooled to −5° C. Asolution of sodium nitrite (155 mg, 2.24 mmol) in water (0.6 mL) wasadded and the reaction mixture was stirred at −5° C. for 20 min, then atroom temperature for 10 min, then at 65° C. for 30 min and finallycooled to room temperature. The reaction mixture was filtered and thesolid was washed with cHCl (2 mL), suspended in MeOH/CH₂Cl₂ (9:1),filtered and dried, to yield the title compound as a solid (125 mg).

Step 4: 5-iodo-1-methyl-3-phenyl-pyrazolo[3,4-c]pyridazin-4-ol

Phenyl[bis(2,2,2-trifluoroacetoxy)]-λ³-iodane (190 mg, 0.43 mmol) wasadded to a suspension of 1-methyl-3-phenyl-pyrazolo[3,4-c]pyridazin-4-ol(166 mg, 0.73 mmol) in CH₂Cl₂ (3.7 mL), followed by the addition ofiodine (111 mg, 0.43 mmol) and pyridine (71 μL). The reaction mixturewas stirred at room temperature for 16 h, then filtered. The collectedsolid was washed with CH₂Cl₂ and dried, to yield the title compound (160mg).

Step 5: 4-chloro-5-iodo-1-methyl-3-phenyl-pyrazolo[3,4-c]pyridazine(Compound IIIa)

5-Iodo-1-methyl-3-phenyl-pyrazolo[3,4-c]pyridazin-4-ol (160 mg, 0.45mmol) in phosphorous oxychloride (0.6 mL) was heated to 120° C. for 10min. The reaction mixture was cooled to room temperature and thesuspension was filtered. The collected solid was dissolved in CH₂Cl₂ andwashed with water. The organic phase was dried (phase separatorcartridge) and concentrated in vacuo, to yield Compound IIIa (130 mg).

¹H NMR δ (ppm)(DMSO-d₆): 7.80-7.76 (2 H, m), 7.62-7.56 (3 H, m),4.43-4.35 (3H, m).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.52 min; m/z 371 [M+H] 97.73% purity.

Example 624-chloro-5-(cyclopenten-1-yl)-1-methyl-3-phenyl-pyrazolo[3,4-c]pyridazine(Compound IIIb)

Nitrogen was bubbled through a suspension of4-chloro-5-iodo-1-methyl-3-phenyl-pyrazolo[3,4-c]pyridazine (60 mg, 0.16mmol), 2-(cyclopenten-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (35mg, 0.18 mmol) and K₃PO₄ (103 mg, 0.48 mmol) in DMF (1 mL) and water(0.3 mL) for 15 min.1,1′-Bis(diphenylphosphino)ferrocene-palladium(dichloridedichloromethane complex (13 mg, 0.016 mmol) was added and the tubesealed and heated to 30° C. for 16 h. The reaction mixture was dilutedwith CH₂Cl₂ and water. The aqueous phase was extracted with CH₂Cl₂ andthe combined organic phases were dried (phase separator cartridge) andconcentrated in vacuo. The resultant residue was purified usingchromatography (silica gel, CH₂Cl₂/isohexane 1:1 to 1:0), to provideCompound IIIb as a solid (10 mg).

¹H NMR δ (ppm) (CHCl₃-d): 7.75-7.69 (2 H, m), 7.52-7.46 (3 H, m),6.62-6.59 (1H, m), 4.39 (3 H, s), 3.11-3.04 (2 H, m), 2.71-2.64 (2 H,m), 2.14-2.04 (2 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 11.75 min; m/z 311 [M+H]93.15% purity.

Example 634-chloro-1-methyl-3-phenyl-5-(3-thienyl)pyrazolo[3,4-c]pyridazine(Compound IIIc)

Compound IIIc was synthesized according to Example 19, but usingN-(2-methyl-5-phenyl-pyrazol-3-yl)acetamide instead ofN-(3-methyl-1H-pyrazol-5-yl)acetamide in Step 2 and using3-ethynylthiophene instead of phenylacetylene in Step 3.

¹H NMR δ (ppm) (CHCl₃-d): 8.01 (1H, dd), 7.79-7.75 (3H, m), 7.53-7.49(3H, m), 7.46 (1H, dd), 4.44 (3H, s).

LCMS (15 cm_Formic_ASCENTIS_HPLC_CH3CN) Rt 10.65 min; m/z 327 [M+H]98.32% purity.

Example 644-chloro-1-methyl-3-phenyl-5-(3-pyridyl)pyrazolo[3,4-c]pyridazine(Compound IIId)

Compound IIId was synthesized according to Example 19, but usingN-(2-methyl-5-phenyl-pyrazol-3-yl)acetamide instead ofN-(3-methyl-1H-pyrazol-5-yl)acetamide in Step 2 and using3-ethynylpyridine instead of phenylacetylene in Step 3.

¹H NMR δ (ppm) (CHCl₃-d): 9.06 (1 H, s), 8.74 (1 H, s), 8.14 (1 H, dt),7.79-7.74 (2H, m), 7.55-7.43 (4 H, m), 4.47 (3 H, s).

LCMS (15 cm_Formic_ASCENTIS_HPLC_CH3CN) Rt 8.99 min; m/z 322 [M+H]98.02% purity.

Example 654-chloro-3-cyclopropyl-1-methyl-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound 21)

Compound 21 was synthesized according to Example 19, but usingN-(5-cyclopropyl-2-methyl-pyrazol-3-yl)acetamide instead ofN-(3-methyl-1H-pyrazol-5-yl)acetamide in Step 2.

¹H NMR δ (ppm) (CHCl₃-d): 7.78 (2 H, m), 7.57-7.47 (3 H, m), 4.26 (3 H,s), 2.61-2.52 (1 H, m), 1.13-1.08 (4 H, m).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.79 min; m/z 285 [M+H] 99.51% purity.

Example 66 4-chloro-1,3-dimethyl-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound 22)

Compound 22 can be synthesized according to Vasilevsky, S. F. andTretyakov, E. V. (1995), “Cinnolines and pyrazolopyridazines: Novelsynthetic and mechanistic aspects of the Richter reaction.”LiebigsAnn./Recl., 1995: 775-779.

¹H NMR δ (ppm) (CHCl₃-d): 7.79-7.75 (2 H, m), 7.56-7.47 (3 H, m), 4.30(3 H, s), 2.80 (3 H, s).

LCMS (10 cm_ESI_Formic_MeCN) Rt 3.69 min; m/z 259 [M+H] 99.32% purity.

Example 67 4-bromo-1,3-dimethyl-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound 23)

Compound 23 can be synthesized according to Vasilevsky, S. F. andTretyakov, E. V. (1995), “Cinnolines and pyrazolopyridazines: Novelsynthetic and mechanistic aspects of the Richter reaction.”LiebigsAnn./Recl., 1995: 775-779.

¹H NMR δ (ppm) (CHCl₃-d): 7.73-7.70 (2 H, m), 7.55-7.46 (3 H, m), 4.30(3 H, s), 2.80 (3 H, s).

LCMS (10 cm_ESI_Formic_MeCN) Rt 3.74 min; m/z 303 [M+H] 99.15% purity.

Example 68 4-fluoro-1,3-dimethyl-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound 24)

A suspension of 4-chloro-1,3-dimethyl-5-phenyl-pyrazolo[3,4-c]pyridazine(44 mg, 0.17 mmol), potassium fluoride (50 mg, 0.85 mmol) in dry DMF (1mL) was stirred at 120° C. for 16 h. The reaction mixture was purifiedby preparative HPLC to provide Compound 24 (12 mg).

¹H NMR δ (ppm) (CHCl₃-d): 8.04-8.00 (2 H, m), 7.58-7.46 (3 H, m), 4.31(3 H, s), 2.75 (3 H, s).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.25 min; m/z 243 [M+H]91.64% purity.

Example 694-chloro-5-(3-fluorophenyl)-1,3-dimethyl-pyrazolo[3,4-c]pyridazine(Compound 25)

Compound 25 was synthesized according to Example 19, but usingN-(2,5-dimethylpyrazol-3-yl)acetamide instead ofN-(3-methyl-1H-pyrazol-5-yl)acetamide in Step 2 and using3-fluorophenylacetylene instead of phenylacetylene in Step 3.

¹H NMR δ (ppm)(CHCl₃-d): 7.59-7.46 (3 H, m), 7.23-7.17 (1 H, m), 4.31 (3H, s), 2.80 (3 H, s).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 4.01 min; m/z 277 [M+H] 99.46% purity.

Example 704-chloro-1-methyl-5-phenyl-3-(3-pyridyl)pyrazolo[3,4-c]pyridazine(Compound

Step 1: (3,5-dichloro-6-phenyl-pyridazin-4-yl)-(3-pyridyl)methanol

To a solution of 4,6-dichloro-3-phenyl-pyridazine (2.27 g, 0.01 mol) indry THF (30 mL) was added pyridine-3-carbaldehyde (1.3 g, 0.012 mol).The reaction mixture was cooled down to −78° C. and a solution of LDA (2N, 22 mL) was added dropwise, keeping the internal temperature below−50° C. The reaction mixture was stirred for 3 h, then water and ethylacetate were added. Phases were separated and the aqueous phase wasextracted with ethyl acetate. The combined organic phases were dried(phase separator cartridge) and concentrated in vacuo. The residue waspurified by column chromatography (silica gel, isohexane/ethyl acetate9:1 to 8:2) yielding the title compound as a solid (4.1 g).

Step 2: (3,5-dichloro-6-phenyl-pyridazin-4-yl)-(3-pyridyl)methanone

A mixture of 3,5-dichloro-6-phenyl-pyridazin-4-yl)-(3-pyridyl)methanol(850 mg, 2.57 mmol) and manganese dioxide (1.1 g, 12.8 mmol) in toluene(20 mL) was stirred at reflux in a Dean-Stark apparatus for 2 h. Thereaction mixture was filtered and the collected solid was washed withCH₂Cl₂. The filtrate was concentrated in vacuo and the residue waspurified by column chromatography (silica gel, isohexane/ethyl acetate8:2) yielding the title compound as a solid (360 mg).

Step 3:4-chloro-1-methyl-5-phenyl-3-(3-pyridyl)pyrazolo[3,4-c]pyridazine(Compound26)

A mixture of (3,5-dichloro-6-phenyl-pyridazin-4-yl)-(3-pyridyl)methanone(100 mg, 0.3 mmol) and methyl hydrazine (19.5 mg, 0.42 mmol) in ethanol(1.5 mL) was stirred in a sealed tube at 60° C. for 3 h. The reactionmixture was concentrated in vacuo and the residue was purified by columnchromatography (silica gel, isohexane/ethyl acetate 9:1 to 8:2) yieldingCompound 26 as a solid (30 mg).

¹H NMR δ (ppm) (CHCl₃-d): 9.05 (1H, m), 8.73 (1H, dd), 8.12 (1H, dt),7.79-7.76 (2H, m), 7.57-7.48 (3H, m), 7.46 (1H, dd), 4.48 (3H, s).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 3.6 min; m/z 322 [M+H] 98.72% purity.

Example 714-chloro-3-cyclopentyl-1-methyl-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound 27)

Compound 27 was synthesized according to Example 70, but usingcyclopentane carboxaldehyde instead of pyridine-3-carbaldehyde in Step1.

¹H NMR δ (ppm) (CHCl₃-d): 7.77 (2 H, m), 7.57-7.47 (3 H, m), 4.30 (3 H,s), 3.87-3.77 (1 H, m), 2.22-2.14 (2H, m), 2.06-1.93 (2 H, m), 1.95-1.83(2 H, m), 1.80-1.71 (2 H, m).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 11.97 min; m/z 313 [M+H]98.14% purity.

Example 72 4-chloro-2-methyl-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound 28)

Step 1: 2-(3-phenyl-1H-pyrazol-5-ylfisoindoline-1,3-dione

A mixture of 3-phenyl-1H-pyrazol-5-amine (6 g, 0.038 mol), phthalicanhydride (5.6 g, 0.038 mol) in acetic acid (60 mL) was heated at 100°C. for 2 h and at 120° C. for 2 h and cooled to room temperature. Thereaction mixture was diluted with water and the suspension was filtered.The collected solid was washed with water, dried, yielding the titlecompound as a solid (10 g).

Step 2: 2-(2-methyl-5-phenyl-pyrazol-3-yl)isoindoline-1,3-dione

Sodium hydride (830 mg, 0.02 mol) was added portionwise to a mixture of2-(3-phenyl-1H-pyrazol-5-yl)isoindoline-1,3-dione (5 g, 0.0173 mol) andmethyl iodide (1.5 mL, 0.024 mol) in DMF (80 mL) at 10° C. The reactionmixture was stirred at room temperature for 16 h. The reaction mixturewas diluted with water and the aqueous phase was extracted with ethylacetate twice. The combined organic phases were dried (MgSO₄), filteredand concentrated in vacuo. The residue was purified by columnchromatography (silica gel, ethyl acetate/isohexane 1:19 to 2:8)yielding a mixture of the title compound and the regioisomer2-(2-methyl-5-phenyl-pyrazol-3-yl)isoindoline-1,3-dione (850 mg, 5:1).

Step 3: 2-(4-iodo-1-methyl-5-phenyl-pyrazol-3-yl)isoindoline-1,3-dione

A suspension of 2-(2-methyl-5-phenyl-pyrazol-3-yl)isoindoline-1,3-dione(850 mg 2.8 mmol), iodic acid (123 mg, 0.7 mmol) and iodine (427 mg,1.68 mmol) in ethanol (30 mL) was heated at 50° C. for 2 h and cooled toroom temperature. The reaction mixture was concentrated in vacuo. Theresidue was partially purified by column chromatography (silica gel,CH₂Cl₂), then triturated with diethyl ether, yielding the title compoundas a solid (660 mg).

Step 4:2-[1-methyl-5-phenyl-4-(2-phenylethynyl)pyrazol-3-yl]isoindoline-1,3-dione

Nitrogen was bubbled through a mixture of2-(4-iodo-1-methyl-5-phenyl-pyrazol-3-yl)isoindoline-1,3-dione (660 mg,1.49 mmol), phenyl acetylene (182 mg, 1.78 mmol), triethylamine (8 mL)and DMF (3 mL) for 15 min. Copper iodide (28 mg, 0.149 mmol) andbis(triphenylphosphine)palladium(II) dichloride (52 g, 0.074 mmol) wereadded and the reaction mixture was stirred at 90° C. under nitrogen for3 h. The reaction mixture was cooled to room temperature, diluted withethyl acetate and water. The organic phase was washed with water andbrine, dried (MgSO₄), filtered and concentrated in vacuo. The residuewas purified by column chromatography (silica gel, isohexane/ethylacetate 8:2 to 6:4) yielding the title compound as a solid (600 mg).

Step 5: 1-methyl-5-phenyl-4-(2-phenylethynyl)pyrazol-3-amine

A mixture of2-[1-methyl-5-phenyl-4-(2-phenylethynyl)pyrazol-3-yl]isoindoline-1,3-dione(300 mg, 0.74 mmol), ethanol (5 mL) and hydrazine hydrate (56 μL, 1.11mL) was stirred in a sealed tube at 90° C. for 1 h and cooled to roomtemperature. The reaction mixture was filtered. The filtrate wasconcentrated in vacuo and purified by column chromatography (silica gel,isohexane/ethyl acetate 7:3 to 1:1) yielding the title compound as asolid (150 mg).

Step 6: 4-chloro-2-methyl-3,5-diphenyl-pyrazolo[3,4-c]pyridazine(Compound 28)

Sodium nitrite (57 mg, 0.82 mmol) was added portionwise to a mixture of1-methyl-5-phenyl-4-(2-phenylethynyl)pyrazol-3-amine (150 mg, 0.55 mmol)in cHCl (5 mL) at 0° C. and the reaction mixture was allowed to warm upto room temperature and stirred for 16 h.

The reaction mixture was poured onto a sodium carbonate solution and theaqueous phase was extracted with ethyl acetate three times. The combinedorganic phases were dried (phase separator cartridge) and concentratedin vacuo. The residue was purified by column chromatography (silica gel,ethyl acetate/isohexane 2:8 to 4:6) yielding Compound 28 as a solid (76mg).

¹H NMR δ (ppm) (CHCl₃-d): 7.84-7.80 (2 H, m), 7.60-7.55 (3 H, m),7.53-7.44 (5 H, m), 4.21 (3 H, s).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.47 min; m/z 321 [M+H] 97.05% purity.

Example 731-[(8-azabicyclo[3.2.1]octan-3-yl]-4-chloro-3-methyl-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound XIIIa)

Compound XIIIa was synthesized from4-chloro-3-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine and nortropinefollowing the general procedure for the Mitsunobu reaction described inExample 20.

¹H NMR δ (ppm) (CHCl₃-d): 8.62 (1 H, s), 7.77 (2 H, m), 7.57-7.48 (3 H,m), 5.62-5.52 (1 H, m), 4.14 (2 H, s), 2.79 (3 H, s), 2.73 (2 H, m),2.32 (2 H, m), 2.19-2.06 (4 H, m).

LCMS (15 cm_Formic_ASCENTIS_HPLC_CH3CN) Rt 7.7 min; m/z 354 [M+H] 93.89%purity.

Example 744-chloro-1-[(3-methylimidazol-4-yl)methyl]-3-(1-methylpyrrol-2-yl)-5-phenyl-pyrazolo[3,4-c]pyridazine(Compound XIIIb)

Compound XIIIb was synthesised following similar procedures of Example 1(Compound Ic), using (1-methyl-1H-imidazol-5-yl)methanol instead of2-(4-methylpiperazin-1-yl)ethanol in Step 7 and1-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrroleinstead of1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inStep 8.

¹H NMR δ (ppm) (CHCl₃-d): 7.76-7.73 (2 H, m), 7.55-7.46 (3 H, m), 7.43(1 H, s), 7.31 (1 H, s), 6.81 (1 H, t), 6.57 (1 H, dd), 6.25 (1 H, dd),5.94 (2 H, s), 3.86 (3 H, s), 3.68 (3 H, s).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 9.81min; m/z 404 [M+H]92.03% purity.

Example 754-chloro-1-methyl-5-phenyl-3-pyrrolidin-1-yl-pyrazolo[3,4-c]pyridazine(Compound XIIIc)

Step 1: 4-chloro-3-iodo-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine

Sodium hydride (60% in mineral oil, 674 mg, 16.9 mmol) was added to asuspension of 4-chloro-3-iodo-5-phenyl-1H-pyrazolo[3,4-c]pyridazine (3g, 8.4 mmol) in dry DMF (42 mL) then methyl iodide (1.05 mL, 16.9 mmol)was added. The reaction mixture was stirred for 2 h. LiCl solution (4%in water) and ethyl acetate were added and the aqueous phase wasextracted with ethyl acetate. The organic phases were combined, dried(MgSO₄), filtered and evaporated. The residue was purified by columnchromatography (silica gel, ethyl acetate/isohexane 0:1 to 1:1) yieldingthe intermediate as a solid (1.51 g).

Step 2: 3-iodo-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-4-ol

A mixture of4-chloro-3-iodo-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine (1.5 g,4.05 mmol) and NaOH aqueous solution (4 M, 2 mL) in DMSO (6 mL) anddioxane (6 mL) was heated to 50° C. for 2.5 h. The mixture was left tocool to rt, then neutralised to pH 2-3, when a precipitate formed. Thesolid was filtered, washed with water and dried, to give 1.33 g of theintermediate.

Step 3:1-methyl-5-phenyl-3-(pyrrolidin-1-yl)-1H-pyrazolo[3,4-c]pyridazin-4-ol

A mixture of 3-iodo-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-4-ol(100 mg, 0.28 mmol), copper iodide (11 mg, 0.056 mmol), L-proline (13mg, 0.11 mmol), K₂CO₃ (193 mmol, 1.4 mmol) in anhydrous DMF (5.6 mL) wasdegassed by bubbling nitrogen through for 10 minutes, then heated to110° C. for 20 h. The mixture was left to cool to room temperature, thenpartitioned between ethyl acetate and water, and the aqueous phase wasextracted with ethyl acetate. The organic phases were combined, dried(MgSO₄), filtered and evaporated. The residue (117 mg) was used as suchin the next step.

Step 4:4-chloro-1-methyl-5-phenyl-3-pyrrolidin-1-yl-pyrazolo[3,4-c]pyridazine(Compound XIIIc)

A suspension of1-methyl-5-phenyl-3-(pyrrolidin-1-yl)-1H-pyrazolo[3,4-c]pyridazin-4-ol(117 mg) in POCl₃ (1.9 mL) was heated to 60° C. for 2.5 h. The mixturewas concentrated in vacuo and the residue was partitioned between CH₂Cl₂and sat. aq. NaHCO₃ solution. The layers were separated and the aqueouswas extracted with CH₂Cl₂, the combined organics were dried (MgSO₄),filtered and concentrated in vacuo. The residue was purified bychromatography on silica gel to give the title compound (81 mg).

¹H NMR δ (ppm) (CHCl₃-d): 7.74-7.70 (2 H, m), 7.53-7.44 (3 H, m), 4.18(3 H, s), 3.62-3.56 (4 H, m), 2.05-1.97 (4 H, m).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.55 min; m/z 314.04 [M+H] 97.21%purity.

Example 764-chloro-3-methyl-5-phenyl-1-(2-pyrrolidin-1-ylethyl)pyrazolo[3,4-c]pyridazine(Compound XIIId)

Compound XIIId was synthesized from4-chloro-3-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine and2-(pyrrolidin-1-yl)ethanol following the general procedure for theMitsunobu reaction described in Example 20.

¹H NMR δ (ppm) (CHCl₃-d): 7.79-7.76 (2 H, m), 7.56-7.47 (3 H, m), 4.82(2 H, t), 3.11 (2 H, t), 2.80 (3 H, s), 2.64 (4 H, m), 1.78-1.72 (4 H,m).

LCMS (10 cm_Formic_ACE_3 C18 AR_HPLC_CH3CN) Rt 9.07 min; m/z 342 [M+H]91.77% purity.

Example 774-chloro-3,5-diphenyl-1-(2-pyrazol-1-ylethyl)pyrazolo[3,4-c]pyridazine(Compound XIVa)

Compound XIVa was synthesized from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine and2-(1H-pyrazol-1-yl)ethanol following the general procedure for theMitsunobu reaction described in Example 20.

¹H NMR δ (ppm) (CHCl₃-d): 7.78-7.69 (4 H, m), 7.55-7.46 (7 H, m), 7.25(1 H, m), 6.17 (1 H, t), 5.25 (2 H, t), 4.87 (2 H, t).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.67 min; m/z 401 [M+H] 99.6% purity.

Example 78 Assay Method Showing Activity of Compounds of the Inventionthat Restore Expression of N48K Clarin-1 (24 hour incubation)

Clarin-1 is the protein encoded by the gene mutated in Usher IIISyndrome (Adato et al., 2002). The most prevalent mutation in Clarin-1in North America is N48K, which is reported to cause loss ofglycosylation and a trafficking defect (Tian et al., 2009). As aconsequence, the N48K protein does not reach the plasma membrane and isdegraded by the proteasome. Thus it is believed that restoring thetrafficking of N48K Clarin-1 to the cell surface provides an avenue ofintervention for Usher III Sydrome.

A useful cellular model to demonstrate the utility of compounds of theinvention that restore expression of N48K Clarin-1 is theHEK293-Clarin-1 N48K-HA D9 cell line (Tian et al., 2009). In a typicalexperiment, these cells were seeded on collagen-coated 96-well plates ata cell density of 20,000 cells per well in Dulbecco's Modified EagleMedium (DMEM) contain 10% fetal bovine serum in a humidified incubatorat 37° C., 5% CO₂. After an overnight incubation, compounds were addedfor a 24 hr incubation in DMEM medium contain 10% fetal bovine serum ina humidified incubator at 37° C., 5% CO₂. As a negative control, DMSOwas used at 0.25% final concentrations. Compounds were typically testedin triplicate fashion. After the 24 hr incubation with compounds, thecells were fixed by the addition of 10% buffered formalin to the wellsto achieve a final concentration of 4% formalin. After a 20 min fixationat room temperature, wells were washed three times withphosphate-buffered saline (PBS) containing Triton X-100 (0.02 phosphate,150 mM NaCl, 0.1% Triton X-100).

The HA-tagged N48K Clarin-1 was detected with an antibody against the HAtag (HA.11 Clone 16B12 Monoclonal antibody, Covance #MMS-101P) at adilution of 1:1000 in PBS containing Triton X-100. After a 90 minincubation, wells were washed three times with PBS containing TritonX-100, and a secondary antibody (Goat anti-mouse IgG-Cy3 (1.5 mg/ml),Jackson IR Europe #115165003) was added to the wells at a dilution of1:250 in PBS containing Triton X-100 for 45 min. Wells were subsequentlywashed three times with PBS containing Triton X-100, and a finalstaining for nuclei was performed by the addition of DAPI(4′,6-diamidino-2-phenylindole) at a dilution of 1:10,000. The imagingof the stained cells was performed on an InCell 1000 High Content Imager(GE Healthcare), reading out the Cy3 channel for N48K Clarin-1 and theDAPI channel for nuclei. The images were analyzed and quantitated usinga specific algorithm. This algorithm measured the HA-Clarin-1 stainingfor each cell based on the additional nuclear segmentation of the DAPIsignal (FIG. 1). This algorithm measured the intensity per cell, andthus it is less sensitive for variation in cell number. Per well,approximately 2,000 cells were measured to achieve an average densityper cell measurement. FIG. 1 illustrates high content analysis.DAPI-stained nuclei are used to perform segmentation of the image intoindividual cells. The density observed in the N48K Clarin-1 channel(Cy3) is then calculated per cell averaged over a field.

Example 79 An Assay Method Showing Activity of Compounds of theInvention that Restore Expression of N48K Clarin-1(2 hour incubation)

Clarin-1 is the protein encoded by the gene mutated in Usher IIISyndrome (Adato et al., 2002). The most prevalent mutation in Clarin-1in North America is N48K, which is reported to cause loss ofglycosylation and a trafficking defect (Tian et al., 2009). As aconsequence, the N48K protein does not reach the plasma membrane and isdegraded by the proteasome. Thus it is believed that restoring thetrafficking of N48K Clarin-1 to the cell surface provides an avenue ofintervention for Usher III Sydrome.

A useful cellular model to demonstrate the utility of compounds of theinvention that restore expression of N48K Clarin-1 is theHEK293-Clarin-1 N48K-HA D9 cell line (Tian et al., 2009). In a typicalexperiment, these cells were seeded on collagen-coated 96-well plates ata cell density of 20,000 cells per well in Dulbecco's Modified EagleMedium (DMEM) contain 10% fetal bovine serum in a humidified incubatorat 37° C., 5% CO₂. After an overnight incubation, compounds were addedfor a 2 hr incubation in DMEM medium contain 10% fetal bovine serum in ahumidified incubator at 37° C., 5% CO₂. As a negative control, DMSO wasused at 0.25% final concentrations. Compounds were typically tested intriplicate fashion. After the 2 hr incubation with compounds, the cellswere incubated in fresh medium for 22 hr. The cells were then fixed bythe addition of 10% buffered formalin to the wells to achieve a finalconcentration of 4% formalin. After a 20 min fixation at roomtemperature, wells were washed three times with phosphate-bufferedsaline (PBS) containing Triton X-100 (0.02 phosphate, 150 mM NaCl, 0.1%Triton X-100).

The HA-tagged N48K Clarin-1 was detected with an antibody against the HAtag (HA.11 Clone 16B12 Monoclonal antibody, Covance #MMS-101P) at adilution of 1:1000 in PBS containing Triton X-100. After a 90 minincubation, wells were washed three times with PBS containing TritonX-100, and a secondary antibody (Goat anti-mouse IgG-Cy3 (1.5 mg/ml),Jackson IR Europe #115165003) was added to the wells at a dilution of1:250 in PBS containing Triton X-100 for 45 min. Wells were subsequentlywashed three times with PBS containing Triton X-100, and a finalstaining for nuclei was performed by the addition of DAPI(4′,6-diamidino-2-phenylindole) at a dilution of 1:10,000. The imagingof the stained cells was performed on an InCell 1000 High Content Imager(GE Healthcare), reading out the Cy3 channel for N48K Clarin-1 and theDAPI channel for nuclei. The images are analyzed and quantitated using aspecific algorithm. This algorithm measured the HA-Clarin-1 staining foreach cell based on the additional nuclear segmentation of the DAPIsignal (FIG. 1). This algorithm measured the intensity per cell, andthus it is less sensitive for variation in cell number. Per well,approximately 2,000 cells were measured to achieve an average densityper cell measurement.

Example 80 IC₅₀ Data for Illustrative Compounds of the Invention

IC₅₀ values for illustrative Pyrazolopyridazine compounds of theinvention were obtained according to the assay method of Example 77.Results are show below in Table 1.

TABLE 1 Compound IC₅₀* Ia A Ib C Ic A Id A Ie A If A Ig A Ih B Ii A Ij AIk A Il A Im A In A Iu B Iv A Iw B Iaa B Ibb B Idd A Iee B Iff A, B IggB Ihh A Iii A IIa A IIb A IIc A IId A IIe A IIf A IIg A IIh A IIi A IIjA IIk A IIl A IIm A IIn A IIo A IIp A IIq A IIr A IIs A IIIc A IIId A 20C 21 B 22 A 23 B 24 A 25 A 26 A 27 B *A = Less than or equal to 2 μM B =Greater than 2 μM and less than or equal to 5 μM C = Greater than 5 μMand less than or equal 21 μM

IC₅₀ values for illustrative Pyrazolopyridazine compounds of theinvention were obtained according to the assay method Example 79.Results are show below in Table 2.

TABLE 2 Compound IC₅₀** Ic E Ii D Ik D Il D Io D Ip E Iq E Ir D Is D ItE Ix F Iy D Iz D Icc D IIa E IIb D IIc D IIf D IIg D IIh D IIj D IIl DIIn D IIq D IIs D IIt D IIu D IIv E IIw E IIx D IIy D IIIa E IIIb EXIIIa E XIIIb E XIIIc F XIIId E XIVa D **D = Less than or equal to 4 μME = Greater than 4 μM and less than or equal to 8 μM F = Greater than 8μM and less than or equal 12 μM

Each reference disclosed in this application is incorporated byreference herein in its entirety.

What is claimed is:
 1. A compound having the structure:

or a pharmaceutically acceptable salt thereof.
 2. A compositioncomprising an effective amount of a compound or pharmaceuticallyacceptable salt of the compound of claim 1 and a pharmaceuticallyacceptable carrier or vehicle.
 3. A method for treating a retinaldegenerative disease, comprising administering to a subject in needthereof an effective amount of a compound or pharmaceutically acceptablesalt of the compound of claim
 1. 4. The method of claim 3, wherein theretinal degenerative disease is retinitis pigmentosa, Leber's congenitalAmaurosis, a syndromic retinal degeneration, age-related maculardegeneration or Usher Syndrome.
 5. A method for treating retinitispigmentosa, Leber's congenial Amaurosis, syndromic retinal degeneration,age-related macular degeneration or Usher Syndrome, comprisingadministering to a subject in need thereof an effective amount of acompound or pharmaceutically acceptable salt of the compound of FormulaI, Formula II, Formula III, Formula XIII, Formula XIV or Formula XV;wherein Formula I has the structure:

wherein R₁ is:

R₂ is:

and a is 0, 1, or 2; wherein Formula II has the structure:

wherein R₃ is:

b is 0 or 1; and c is 1 or 2; wherein Formula III has the structure:

wherein R₄ is

wherein Formula XIII has the structure:

wherein R₅ is:

R₆ is:

 and a is 0, 1, or 2; wherein Formula XIV has the structure:

wherein R₇ is:

b is 0 or 1; and c is 1 or 2; and wherein Formula XV has the structure:

wherein R₈ is:


6. The method of claim 5, wherein the Usher syndrome is Usher ISyndrome, Usher II Syndrome, or Usher III Syndrome.
 7. The method ofclaim 5, wherein the Usher syndrome is Usher III syndrome.
 8. A methodfor treating retinitis pigmentosa, Leber's congenital oAmaurosis,syndromic retinal degeneration, age-related macular degeneration orUsher Syndrome, comprising administering to a subject in need thereof aneffective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof, or XIIIa:


9. The method of claim 8, wherein the Usher syndrome is Usher ISyndrome, Usher II Syndrome, or Usher III Syndrome.
 10. The method ofclaim 8, wherein the Usher syndrome is Usher III syndrome.
 11. Themethod of claim 4, wherein the retinal degenerative disease is UsherSyndrome.
 12. A method for treating hearing loss associated with Ushersyndrome, comprising administering to a subject in need thereof aneffective amount of a compound or pharmaceutically acceptable salt ofthe compound of claim
 1. 13. The method of claim 12, wherein the Ushersyndrome is Usher I Syndrome, Usher II Syndrome, or Usher III Syndrome.14. The method of claim 12, wherein the Usher syndrome is Usher IIIsyndrome.
 15. The method of claim 11, wherein the Usher syndrome isUsher I Syndrome, Usher II Syndrome, or Usher III Syndrome.
 16. Themethod of claim 11, wherein the Usher syndrome is Usher III syndrome.